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. Overall design: 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.

基孔肯雅病毒（Chikungunya virus, CHIKV）属于甲病毒属（Alphavirus），经伊蚊（Aedes）传播，可使哺乳动物出现发热、皮疹与关节痛症状。目前，CHIKV非结构蛋白3（non-structural protein 3, nsP3）的功能仍尚不明确。本研究在既往研究基础上，针对nsP3甲病毒特有结构域（alphavirus unique domain, AUD）内的保守且暴露于表面的簇区域构建了一组突变体，并利用亚基因组复制子（sub-genomic replicon, SGR）对其复制能力进行检测。结果显示，其中3株SGR突变体可在伊蚊来源细胞中高效复制，但在哺乳动物细胞中复制能力显著减弱。进一步研究发现，该复制差异源于细胞培养温度差异（伊蚊细胞培养温度为28℃；哺乳动物细胞为37℃）：当哺乳动物细胞在亚生理温度（28℃）下培养时，上述突变体未出现复制缺陷。该现象在感染性CHIKV及亲缘关系密切的奥尼永-尼永病毒（O'nyong-nyong virus）中均得到验证。值得注意的是，野生型SGR在28℃下的复制效率显著高于37℃。

为探究该差异背后的机制，本研究聚焦于两条已知的抗病毒通路：干扰素刺激基因（interferon-stimulated genes, ISGs）通路与应激颗粒（stress granules, SG）通路。分析发现，在37℃下，SGR的复制与ISGs的表达上调呈显著正相关；而在28℃下，二者相关性较弱。同时，我们观察到在28℃条件下，应激颗粒蛋白（G3BP、eIF4G及TIA-1）向基因组复制的细胞质位点的招募作用增强。

上述发现具有实际应用价值：当伊蚊叮咬哺乳动物时，病毒首先感染外周组织细胞，而此类组织的温度通常处于亚生理水平。我们推测，包括CHIKV在内的甲病毒已进化出相应机制，可在这类细胞中同时促进病毒基因组复制并抑制抗病毒免疫应答。

整体实验设计：将转染了CHIKV-D-SGR野生型、GAA突变体或空白对照的C2C12细胞分别置于37℃与28℃下培养12 h或24 h，随后进行比较基因表达谱测序（RNA-seq）。