@article {Guo2021.09.29.462334, author = {Jinchao Guo and Mark Harris}, title = {Enhancement of Chikungunya virus genome replication in mammalian cells at sub-physiological temperatures}, elocation-id = {2021.09.29.462334}, year = {2022}, doi = {10.1101/2021.09.29.462334}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Chikungunya virus (CHIKV) is an Alphavirus transmitted by Aedes mosquitoes, causing fever, rash and arthralgia. The function of the CHIKV non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019), we generated a panel of mutants in a conserved and surface-exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication phenotype using a sub-genomic replicon (SGR) in mammalian and mosquito cells. Three mutants that replicated poorly in mammalian cells showed no defect in mosquito cells. These mutants were temperature-sensitive, rather than species-specific, as they exhibited no replication defect in mammalian cells at sub-physiological temperature (28{\textdegree}C). Similar effects were observed in the context of infectious CHIKV and the closely related O{\textquoteright}Nyong Nyong virus. This analysis also revealed that the wildtype SGR replicated more efficiently at 28{\textdegree}C compared to 37{\textdegree}C. This was not due to either impaired interferon responses as the enhancement was observed in Vero cells, or to a defect in eIF2α phosphorylation as treatment with ISRIB, an inhibitor of global translation attenuation, did not compensate for replication defects at 37{\textdegree}C. The phenotype did correlate with enhanced recruitment of stress granule proteins (G3BP, eIF4G and TIA-1) into cytoplasmic sites of genome replication at 28{\textdegree}C. As cells in the periphery will be at sub-physiological temperatures, and will be the first cells infected in the mammalian host following a mosquito bite, we propose that alphaviruses such as CHIKV have evolved mechanisms to both promote viral genome replication and concomitantly limit antiviral responses in these cells.Importance Chikungunya virus (CHIKV) is a re-emerging arbovirus, transmitted by Aedes mosquitos and posing epidemic threats. Arboviruses must be able to replicate efficiently in both the mosquito vector and the mammalian host, at different temperatures. Following a mosquito bite the first cells infected will be in the skin and at sub-physiological temperature (less than 37{\textdegree}C). We show that mutants in one of the CHIKV proteins (nsP3) could not replicate at 37{\textdegree}C, but replicated efficiently in mammalian cells at 28{\textdegree}C. We also showed that wildtype CHIKV replicated more efficiently at 28{\textdegree}C in comparison to 37{\textdegree}C in mammalian cells. We investigated the mechanism behind this observation and showed that at sub-physiological temperatures proteins present in cytoplasmic stress granules were more efficiently recruited to sites of virus replication. We propose that CHIKV has evolved mechanisms to promote their replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a mosquito bite.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2022/08/03/2021.09.29.462334}, eprint = {https://www.biorxiv.org/content/early/2022/08/03/2021.09.29.462334.full.pdf}, journal = {bioRxiv} }