The genus Flavivirus consist of several clinically important viruses such as Yellow Fever virus, Dengue virus, Japanese Encephalitis virus, Tick-borne Encephalitis virus and West Nile virus (WNV). The Flavivirus non-structural protein 2A (NS2A) has been shown to be a multifunctional protein involved in virion packaging, RNA replication, and modulation of IFN response. Given the importance of NS2A in the virus life cycle, and also its involvement in modulating the host innate immune response, it is of interest to define the NS2A-host protein interaction profile. This however has been hindered by the lack of specific antibodies against NS2A due to its highly membrane-associated structure.
This project is aimed at generating and characterising an infectious virus with an epitope-tagged WNV NS2A, to allow the discovery of NS2A interacting partners. We report here the insertion of a HA-tag peptide (YPYDVPDYA) inserted into various location (aa position: 69, 100,131, 167, 168, 192 and 227) within NS2A. Thereafter, serial passing of the tagged viruses will be performed to determine the stability of the tag and replication efficiency of the tagged viruses. Using the most stably tagged variant, NS2A co-immunoprecipitation studies and aa mutations in NS2A will be further characterised to determine their mechanistic functions in the virus life cycle.