Enhancement of Chikungunya virus genome replication in mammalian cells at sub-physiological temperatures

Chikungunya virus (CHIKV) is an Alphavirus transmitted by Aedes mosquitoes, causing fever, rash and arthralgia in mammals. The function of the CHIKV non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies, we generated a panel of mutants in a conserved and surface-exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication using a sub-genomic replicon (SGR). Three of these SGR mutants replicated well in mosquito cells but poorly in mammalian cells. We observed that this difference was due to cell culture temperature (mosquito cells: 28 °C; mammalian cells: 37 °C) as the mutants exhibited no replication defect in mammalian cells grown at a sub-physiological temperature (28 °C). Similar effects were observed for infectious CHIKV and the closely related O'nyong-nyong virus. Intriguingly, the wildtype SGR replicated more efficiently in mammalian cells at 28 °C compared to 37 °C. To explore the mechanism behind this difference, we focused on two known antiviral pathways: interferon-stimulated genes (ISGs) and stress granules (SG). SGR replication correlated strongly with increased expression of ISGs at 37°C, but only weakly correlated at 28 °C. We also observed enhanced recruitment of SG proteins (G3BP, eIF4G, and TIA-1) into cytoplasmic sites of genome replication at 28 °C. These findings may have real-world implications as when a mosquito bites a mammal, the virus first infects cells in peripheral tissues which are often at sub-physiological temperatures. We propose that alphaviruses such as CHIKV have evolved mechanisms to both promote viral genome replication and concomitantly limit antiviral responses in these cells. Comparative gene expression profiling (RNA-seq) of C2C12 cells transfected with CHIKV-D-SGR WT, GAA, or mock, incubated at 37C or 28 °C, for 12 h or 24 h.