Molecular and Fitness Barriers to Bunyavirus Reassortment

Project Summary / Abstract The bunyavirus order (Bunyavirales) includes significant human, animal, and plant pathogens. As with all segmented viruses, reassortment is a major driver of bunyavirus evolution. Reassortment can produce viruses with undesirable phenotypes, including the ability to infect new hosts. As the world becomes increasingly interconnected and disrupted by climate change, the opportunity for previously isolated bunyaviruses to meet and reassort is increasing. Compatibility between the proteins and RNAs of two viruses is a key determinant of whether they can produce viable reassortant progeny. And, because reassortment joins proteins and RNAs that have not adapted to work together, new reassortants face an uphill evolutionary battle when competing with their parents and other viruses in the population. This proposal will generate an improved mechanistic understanding of molecular barriers to reassortment and investigate evolutionary pathways that permit reassortants to emerge despite initial fitness disadvantages. We have devised a new system that uses libraries of competing minigenomes to quantify reassortment potential between large numbers of viruses simultaneously, and to define the molecular breakdown in cases when they can't. Using these "minigenome melees" in concert with traditional techniques, we propose to answer targeted questions about the molecular compatibility between bunyaviruses. Our team combines expertise in viral genomics, molecular virology, mosquito infection, and virus evolution. In aim 1, we identify steps in the viral lifecycle that break down when bunyavirus replication proteins are mismatched and perform directed evolution experiments that force mismatched proteins to adapt to work together. This will explain a key constraint on bunyavirus reassortment and detail molecular interactions at the heart of the bunyavirus life cycle. In aim 2, we use our minigenome melee system to test the hypothesis that orthobunyavirus reassortment is relatively unconstrained by packaging. In aim 3, we test the hypothesis that bottlenecks associated with replication in mosquitoes enable less fit reassortant genotypes to gain a foothold in populations via the stochastic effect of genetic drift. At the conclusion of this project, we expect to have a substantially improved understanding of the molecular rules that determine whether two bunyaviruses can reassort, the fitness consequences of reassortment, and the evolutionary pathways by which reassortant viruses emerge. Our results could be used to parameterize models that predict bunyavirus emergence risk and will shed light on conserved interactions that could be targeted by antiviral drugs. Minigenome melees could in principle be used to study the biology and evolution of all kinds of RNA viruses, and we expect this work to establish this as a broadly useful platform.