Unravelling a co-nsP-iracy: the role of chikungunya virus non-structural protein 3 in replication and pathogenesis

Chikungunya virus (CHIKV) is a member of the Alphavirus genus, transmitted to humans by mosquitoes of the Aedesgenera. Infection with CHIKV causes chikungunya fever, which in many cases can lead to chronic joint disease, leaving patients with reduced ambulation. Despite its rising potential as a threat to global health, no effective vaccine or antiviral agent for protection or treatment are available. The CHIKV non-structural protein 3 (nsP3) is essential to the virus lifecycle and is believed to be a component of the genome replication complex. However, to date, the exact role of this protein has yet been determined. Although a conserved polyproline motif in the C-terminal hypervariable domain of nsP3 has been reported to interact with cellular SH3 domains, the function of this motif remains enigmatic. To address this question we generated a panel of mutations in this motif and tested the phenotype in the context of both a subgenomic replicon and full-length infectious virus, in both mammalian and mosquito-derived cell lines. Most of the mutations were well tolerated in the sub-genomic replicon, however, a subset either attenuated or completely abolished production of infectious CHIKV. These results suggest that as well as its role in genome replication, nsP3 also functions during assembly and release of infectious virus particles and that the C-terminal polyproline motif is a critical determinant of this function.