Defining the temporal roles of viral and cellular protein interactions during enterovirus replication

Despite decades of research to uncover the complete composition of the picornavirus translation machinery and RNA replication complex, the exact protein components of these vital parts of the virus life cycle remain unknown. Over the course of infection, viral RNA must serve as a template for translation, RNA synthesis, and ultimately encapsidation into progeny virions. Picornaviruses direct the different processes of viral replication through interactions of RNA elements in the genomic RNA with both cellular and viral proteins; therefore, each step of the viral life cycle requires a unique and specific ribonucleoprotein (RNP) complex. These small, positive-strand RNA viruses capitalize on their small genome size by utilizing multifunctional products of the viral polyprotein, which are necessary for RNA replication. Adding to the complexity of the composition of viral RNA replication complexes is the appropriation of cellular proteins for use in viral functions. This thesis aims to address viral and cellular protein interactions necessary for each stage of the RNA replication cycle through investigations of both protein-protein and RNA-protein interactions.;Past efforts to isolate the poliovirus RNA replication complex have relied on crude purifications through fractionation or sequential centrifugation; while these methods successfully enriched for RNA synthesis activity, they contained all viral proteins indicating a lack of highly purified complexes. In Chapter 2 of this thesis, we attempted to purify the RNA replication complex through a gentle purification procedure to isolate exogenously expressed poliovirus FLAG-His tagged 3CD from infected cells or from in vitro translation/replication reactions. The viral proteinase 3CD plays an essential role in negative-strand RNA synthesis initiation by its interaction at the 5' stem-loop I RNA element in positive-strand to form a ternary complex with cellular protein poly(rC) binding protein 2 (PCBP2), as well as by stimulating the uridylylation of the protein primer for replication (VPg) by the viral polymerase (3D). We were unable to detect changes in viral protein interactions with 3CD and predict that a more sensitive assay is necessary. An alternative, RNA-affinity based approach to isolate viral RNP complexes using a poliovirus mutant containing RNA-affinity tags is described in Chapter 3. Through RNA-affinity purification of viral RNA, we were able to confirm the interaction of specific cellular proteins with viral RNA during infection; however, current purifications were unable to identify specific RNP complex components over background.;In addition to viral and cellular proteins that aid in viral replication, viral RNA is also subject to interaction with restriction factors of viral infection. We further defined one such interaction in Chapter 4: the binding of mRNA decay factor AU-rich binding factor 1 (AUF1) to the highly structured 5' noncoding region of poliovirus RNA. We explored viral evasion of this negative regulation, both by viral proteinase cleavage of AUF1 and spatial sequestration of this protein to the periphery of the cytoplasm. Models for both the formation of viral ribonucleoprotein complexes and viral evasion of negative regulation are discussed in Chapter 5. Overall, this thesis demonstrates progress towards isolating viral RNP complexes and provides details for interactions of restriction factors with the viral RNA.