Chikungunya virus (CHIKV) is a re-emerging alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). nsP3 consists of three domains, of these the macrodomain is reported to have ADP-ribose and RNA-binding activity, the hypervariable region is involved in various interaction with host proteins, however, the alphavirus unique domain (AUD), as a homologous sequence unique to alphaviruses, is essential for CHIKV replication with absolutely unknown functions.
To investigate the function of AUD, a mutagenic analysis was performed. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutant panel was also analysed in the context of infectious CHIKV. Results indicated that, in addition to a role in RNA replication, the AUD was also required for virus assembly.
Further analysis revealed that one mutant (P247A/V248A) specifically blocked transcription of the subgenomic RNA leading to a dramatic reduction in synthesis of the structural proteins and concomitant reduction in virus production. This phenotype could be explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. A high-resolution confocal microscopy analysis on the track of nsP3, capsid protein and dsRNA confirmed the P247A/V248A replication defect.
In parallel, this project also set out to investigate a variety of biochemical characters of nsP3/AUD, for example, RNAi suppression activity, self-multimerization and interactions with cellular proteins by the approach of quantitative proteomic analysis. In conclusion, this study reveals that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.
