Elucidating the mechanisms of viral life cycles under near-native conditions

Project Summary Mononegavirales is a taxonomic order of viruses, so classified for their negative sense single stranded RNA genome and their pleomorphic membrane-enveloped virions. Mononegavirus life cycles involve a number of different events, including entry of virions into host cells, viral mRNA transcription, genome replication and virus assembly, and viral budding from host cells. These events are carried out through molecular interactions between virus and host cell machinery; elucidating these interactions is key to understanding viral life cycles and identifying potential therapeutic targets. Studies of viral machinery are typically limited to isolated particles or assemblies; this removes them from their native environments and strips away important molecular interactions. To preserve biological context, viral machinery must be studied in situ, i.e. under near-native conditions, such as within intact virions or cells. These environments are a complex, disordered mixture of molecules, making it particularly difficult to obtain molecular resolution information. Here, we propose to study three mononegaviruses: measles, rabies, and Ebola viruses. Each serve as prototypical viruses for their taxonomic families, and each are pathogens important to global health. To carry out our proposed research, we will use and develop in situ structural biology methods. Our primary method will be cryo-electron tomography (cryo-ET), a type of cryo-electron microscopy (cryo-EM) that allows for visualization of three-dimensional volumes. This overcomes the typical cryo-EM requirement of thin monolayers of purified particles, allowing for the acquisition of molecular-resolution information in near-native environments. We will develop data collection and computational methods for cryo-ET to enable rapid, automated data collection, high-resolution structure determination, and accurate molecular identification. We will also use and develop methods complementary to cryo-ET including focused ion-beam milling and correlative light and electron microscopy approaches. Our research will provide novel biological insights three important viruses, but more broadly, it will demonstrate a transformative approach for studying viruses. Rather than trying to tease apart function and interactions through indirect biochemical means, our research will provide an infrastructure to directly observe the virus and host cell machinery with complete biological contexts under near-native conditions.