Mechanistic Constraints on the Acquisition of Variation by Alphaviruses

This project asks: Does the genome structure of RNA viruses place mechanistic constraints on acquisition of variation by the virus; and can these be defined in a way that aids the design of data handling and analysis algorithms for metasequencing applications? It addresses the BBSRC Theme 'Exploiting New Ways of Working': Strategic Priority 'Data Driven Biology'; Investigating links between phenotypic traits and variation in biological systems or processes. RNA polymerases of RNA viruses are error prone, introducing mutations at much higher rates than the proof-reading DNA polymerases of their hosts. The natural replication of RNA virus genomes is a potent source of genetic variation upon which both natural and artificial selection may act. Several notable emerging diseases are caused by RNA viruses. Their emergence has been attributed partly to the ability to evolve rapidly, but there is little quantitative data on what may constrain this. The high virulence of some RNA viruses has also led to concerns regarding intentional misuse. Consequently a greater understanding of viral evolution is required to help predict and understand emergence events, as well as to detect and distinguish natural and synthetic organisms rapidly and reliably. Studies of RNA viruses have demonstrated that codon usage can vary along the length of a polycistronic RNA, and this correlates with sites of co-translational modification. This may be an adaptive mechanism to slow the rate of translation at points along the polycistron to facilitate co-translational modification, and may represent a constraint on variability. Furthermore, some studies have connected virus RNA secondary structure with immune response induction, which may result in selection on the basis of genome structure. Beyond this, it is not clear what constraints on the acquisition of variation in RNA viruses apply, except those associated with non-silent mutations. DNA sequencing now allows metasequencing of complex biological milieux comprising multiple taxa, and identification of distinct genomes through data handling algorithms. The data handling challenges of metasequencing could be streamlined by algorithms to rank potential genomes during assembly, giving priority to those with a greater likelihood of being real. They could also be streamlined by algorithms to rapidly identify synthetic genomes in predominantly natural milieux. The project will examine the ability of a synthetic Hazard Group 2 alphavirus genome to evolve towards an optimal sequence, potentially passing through an intermediate that is less optimal than the initial modified form. It will compare wildtype and computationally optimized sequences; and the stability and direction-of-mutation of hybrid sequences. The project will produce quantitative data on the mutability of RNA viruses and factors which militate against variation, providing data for the future optimization of algorithms for sifting metasequence data to rapidly and reliably identify synthetic organisms; and for rapid identification and classification of previously unknown organisms, and emergent or potential pathogens in complex biological mixtures. The results will have applications in evolutionary biology, environmental
monitoring, epidemiology, forensic science, and medical and veterinary diagnostics. The project combines Pirbright's expertise with recombinant alphaviruses with University Of Surrey's interest in genetic and phenotypic variation in zoonotic RNA viruses, and Dstl's interest in the dynamics of alphavirus quasispecies. The project will expand TPI's portfolio on the cell and molecular biology of alphaviruses; it will inform UoS' analyses of zoonotic viruses; and expand Dstl's portfolio with CL2 surrogates for biothreat viruses that help to understand the population dynamics of RNA viruses generally. It will begin to assess potential limits on the nature of threat from both nature of threat from both natural & synthetic sources.