Establishing a model system to study CHIKV nsP4 and its host interactome during genome replication and translation

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus, that causes large outbreaks that have high morbidity, due to arthralgia symptoms associated with chronic pain. CHIKV encodes four non-structural proteins (nsPs), including the RNA dependent RNA polymerase nsP4. Interactions of nsP4 with host cell proteins are poorly categorised, due to its rapid degradation in vivo and the insolubility of recombinantly expressed protein.
The aim of this study was to investigate interactions between nsP4 and host cell proteins during CHIKV replication and translation, using a sub-genomic replicon (SGR) system that encodes CHIKV nsPs but has a luciferase reporter gene in place of the structural viral proteins. Attempts to optimise an antibody against nsP4 for use in co-immunoprecipitation and quantitative mass spectrometry were hindered by the unreliable and unspecific nature of available nsP4 antibodies. Consequently, this study went on to develop a novel system to study nsP4 interactions in a CHIKV SGR expressing recombinant nsP4, labelled with N-terminal FLAG tags (termed nsP4-3XF SGR).
nsP4-3XF SGR was successfully engineered by cloning the sequence of a tagged nsP4 from an existing recombinant virus into the SGR, which was then validated for replication efficiency and nsP4-3XF expression. Expression of nsP4-3XF decreased the level of SGR replication in comparison to the wildtype SGR, but replicated to a sufficient level that it could be used for further study of nsP4 interactions. Western blotting demonstrated that nsP4-3XF could be detected in transfected BHK-21 cell lysates but not in RD, Huh7 or C6/36 cells. Preliminary co-immunoprecipitation assays from transfected BHK-21 cells confirmed purification of nsP4-3XF in complex with host cell proteins HSP90 and TMEM45B which have previously been described as interactors of CHIKV nsP4. As such, the preliminary data demonstrated that nsP4-3XF SGR, engineered and validated by this study will be an invaluable system for studying the CHIKV nsP4 proteome complex during CHIKV replication and translation.