Many flaviviruses are adapted to dual-host transmission and maintained in cycles between hematophagous arthropods (i.e. mosquitoes and ticks) and vertebrates. An example of mosquito-borne flavivirus (MBFV) is Zika virus (ZIKV), which is a human pathogen of global concern. Other flaviviruses, such as Long Pine Key virus (LPKV), replicate in mosquitoes and phylogenetically affiliate with MBFVs, but lack the capacity to infect vertebrate cells. These viruses are known as dual-host affiliated insect-specific flaviviruses (dISFs). The precise sequence elements and virus-host interactions that modulate the differential host ranges of MBFVs and dISFs have not been defined. Identification of these sequences and virus-host interactions would provide key insight into why some flaviviruses infect and cause devastating disease in humans while others are insect-specific. In the initial funding period, we identified the broad genetic determinants that modulate the differential host ranges of MBFVs and dISFs. Through the construction and characterization of chimeric viruses, we demonstrated that ZIKV loses its vertebrate-infecting tropism when its 5’ untranslated region (UTR), adjacent capsid protein (C) gene, and 3’ UTR are replaced with those of LPKV, while its mosquito-infecting phenotype is retained. The UTRs contain highly structured elements that interact with NS3 (the viral helicase) and NS5 (the viral RNA polymerase) to regulate genome replicate. The UTRs, NS3, and NS5 of MBFVs also interact with many host proteins, but the host proteins that comprise the dISF replication complex are unknown. The overall goal of this continuation is to dissect the 5’- and 3’-terminal ends of the flavivirus genome to pinpoint the precise sequences responsible for flavivirus host-specificity and to compare the virus-host interactions that occur during MBFV and dISF replication through the identification and functional characterization of host proteins that bind to their UTRs, NS3, and NS5. Three independent aims have been designed to achieve this goal. In aim 1, we will dissect the 5’ UTR, C gene, and 3’ UTR of the genomes of LPKV and ZIKV to identify the specific sequences responsible for their differential host ranges. Chimeras of LPKV and ZIKV will be created and the abilities of the resulting viruses to replicate in vertebrate and mosquito cells will be assessed. We will initially focus on each secondary structure (e.g. stem-loops, hairpins, dumbbells), either alone or together with others, then target specific sequences in structures of greatest interest. Subsequent experiments will be performed using additional dISFs and MBFVs to determine whether the genetic determinants that modulate host-specificity are shared among flaviviruses of the same group. In aim 2, we will identify and functionally characterize host proteins that bind to the 5’- and 3’-terminal ends of the dISF and MBFV genomes. Biotinylated RNAs containing the 5’ UTR, C gene, and 3’ UTR sequences of select dISFs and MBFVs will be transfected into mosquito and vertebrate cells. The cell cultures will be inoculated with virus then RNA-binding proteins (RBPs) will be recovered using streptavidin beads and identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. We will pinpoint the precise viral sequences that interact with RBPs of greatest interest by reversible RNA-protein crosslinking, immunoprecipitation, and RNAseq. Select RBPs will be further analyzed in overexpression and knockdown experiments to determine whether they are positive or negative regulators of viral replication. In aim 3, we will identify and functionally characterize host proteins that bind to NS3 and NS5 of select dISFs and MBFVs. A novel proximity-based protein labeling technique known as TurboID will be used to recover mosquito and vertebrate hosts that associate, either directly or indirectly, to NS3 and NS5 of the selected viruses. Recovered proteins will be identified by LC-MS/MS analysis and a subset will be characterized in overexpression and knockdown experiments to determine whether they enhance or suppress viral replication. Data generated from the experiments outlined in the aforementioned aims will allow us to define the genetic and molecular determinants of flavivirus host-specificity.

This project will identify the precise genomic determinants and molecular mechanisms that condition the differential host ranges of insect-specific and mosquito/vertebrate flaviviruses, providing information that will assist in the rationale design of novel intervention strategies to control flavivirus transmission and disease.