Enhancement of alphavirus 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 (Gao et al, 2019) [1], 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 culture temperature (mosquito cells: 28{degrees}C; mammalian cells: 37{degrees}C), as the mutants exhibited no replication defect in mammalian cells grown at a sub-physiological temperature (28{degrees}C). Similar phenotypes were observed for infectious CHIKV and the closely related ONyong Nyong virus. Intriguingly, the wildtype SGR replicated more efficiently in mammalian cells at 28{degrees}C compared to 37{degrees}C. To explore the mechanism behind this difference, we focused on two known antiviral pathways: interferon-stimulated genes (ISGs) and stress granules (SGs). SGR replication was concomitant with increased expression of ISGs at 37{degrees}C, but this response was impaired at 28{degrees}C. We also observed enhanced recruitment of the SG component G3BP1 into cytoplasmic sites of viral genome replication at 28{degrees}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. Significance statementChikungunya virus (CHIKV) is a re-emerging arbovirus that is transmitted by Aedes mosquitos and poses epidemic threats. Arboviruses must be able to grow efficiently in both the mosquito vector and the mammalian host, which have different body temperatures. Following a mosquito bite, the first cells infected are typically in the skin and are at sub-physiological temperatures, ie lower than the internal body temperature (37{degrees}C). Here we show that CHIKV grows more efficiently in mammalian cells cultured at 28{degrees}C, compared to 37{degrees}C. We investigated the mechanism behind this increase in growth efficiency and demonstrated that at the lower temperature the production of antiviral genes was impaired. Additionally, proteins that respond to cellular stress and are needed for virus replication were more abundantly recruited to sites in the cell where the virus was replicating. We propose that CHIKV has evolved mechanisms to promote its replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a mosquito bite.