Infection by dengue virus is a growing concern due to lack of therapeutic or prophylactic measures to combat dengue infection, and the expanding habitat of the mosquito vector responsible for transmission. Like other viruses, dengue virus uses physical interactions with host proteins to co-opt their function and accomplish the numerous tasks required for replication, and a deeper understanding of how dengue virus hijacks host machinery will be useful for therapeutic development. We aim genetic approaches to gain a mechanistic understanding of dengue-host functional relationships and link them to the underlying physical interactions that we previously identified.
Viruses interact with the host to orchestrate myriad biochemical processes required for replication. A unique characteristic of arboviruses such as dengue virus (DENV), which are transmitted to vertebrates by arthropod vectors, is that these viruses must maintain interactions in two evolutionarily distant hosts to successfully replicate. I this proposal, comparative genetic interaction mapping will be used to identify conserved host factors that are functionally important for DENV replication:
Aim 1-Quantitatively map evolutionary constraints of DENV-host interactions A genetic interaction between two factors implies that they are functionally related. Single and pairwise RNAi knockdown coupled with a reporter virus system will be used to quantitatively map genetic interactions of host factors that physically interact with DENV. This will be the first systematic study of genetic interactions in a host-pathogen system. Hierarchical clustering will be applied to the data generated in Aim 1 to identify host factors with similar genetic interaction profiles. Previously acquired DENV-host and host-host protein interaction data will be incorporated to define functional modules. Enrichment analysis will be used to identify functional groups and pathways that are enriched for host replication or restriction factors. The results from Aim 1 will address fundamental questions regarding the evolutionary constraints imposed by hosts on DENV replication.
Aim 2-Identify the point of defect in the viral replication cycle for a subset of host replication factors from conserved functional modules Viral replication will be measured at the point of translation, RNA synthesis and exit in both host systems following knockdown by RNAi for 10 host replication factors from conserved functional modules characterized in Aim 1. The experiments in this aim will provide insight into the molecular mechanism of DENV replication in two evolutionarily distant hosts. Ultimately, an evolutionary perspective on DENV-host interactions will be particularly useful for drug development. Targeting conserved or evolutionarily constrained interactions will yield more robust therapies, as DENV will be limited in its ability to evolve resistance.