Department of Molecular Microbiology and Center for Women Infectious Disease Research, Washington University School of Medicine in St. Louis, Missouri, United States of America.
PLoS Biol. 2023 Nov 20;21(11):e3002381. doi: 10.1371/journal.pbio.3002381. eCollection 2023 Nov.
Antiviral responses are often accompanied by translation inhibition and formation of stress granules (SGs) in infected cells. However, the triggers for these processes and their role during infection remain subjects of active investigation. Copy-back viral genomes (cbVGs) are the primary inducers of the mitochondrial antiviral signaling (MAVS) pathway and antiviral immunity during Sendai virus (SeV) and respiratory syncytial virus (RSV) infections. The relationship between cbVGs and cellular stress during viral infections is unknown. Here, we show that SGs form during infections containing high levels of cbVGs, and not during infections with low levels of cbVGs. Moreover, using RNA fluorescent in situ hybridization to differentiate accumulation of standard viral genomes from cbVGs at a single-cell level during infection, we show that SGs form exclusively in cells that accumulate high levels of cbVGs. Protein kinase R (PKR) activation is increased during high cbVG infections and, as expected, is necessary for virus-induced SGs. However, SGs form independent of MAVS signaling, demonstrating that cbVGs induce antiviral immunity and SG formation through 2 independent mechanisms. Furthermore, we show that translation inhibition and SG formation do not affect the overall expression of interferon and interferon stimulated genes during infection, making the stress response dispensable for global antiviral immunity. Using live-cell imaging, we show that SG formation is highly dynamic and correlates with a drastic reduction of viral protein expression even in cells infected for several days. Through analysis of active protein translation at a single-cell level, we show that infected cells that form SGs show inhibition of protein translation. Together, our data reveal a new cbVG-driven mechanism of viral interference where cbVGs induce PKR-mediated translation inhibition and SG formation, leading to a reduction in viral protein expression without altering overall antiviral immunity.
抗病毒反应通常伴随着翻译抑制和应激颗粒 (SGs) 的形成感染细胞。然而，这些过程的触发因素及其在感染过程中的作用仍然是研究的热点。复制后病毒基因组 (cbVGs) 是仙台病毒 (SeV) 和呼吸道合胞病毒 (RSV) 感染期间线粒体抗病毒信号 (MAVS) 途径和抗病毒免疫的主要诱导物。cbVGs 与病毒感染期间细胞应激之间的关系尚不清楚。在这里，我们表明，在含有高水平 cbVGs 的感染中会形成 SGs，而在含有低水平 cbVGs 的感染中则不会形成 SGs。此外，我们使用 RNA 荧光原位杂交技术在感染过程中以单细胞水平区分标准病毒基因组和 cbVGs 的积累，表明 SGs 仅在积累高水平 cbVGs 的细胞中形成。PKR 激活在高 cbVG 感染期间增加，并且如预期的那样，是病毒诱导的 SGs 所必需的。然而，SGs 的形成独立于 MAVS 信号，表明 cbVGs 通过 2 种独立的机制诱导抗病毒免疫和 SG 形成。此外，我们表明，翻译抑制和 SG 形成不会影响感染过程中干扰素和干扰素刺激基因的总体表达，从而使应激反应对于全局抗病毒免疫不是必需的。通过活细胞成像，我们表明 SG 的形成是高度动态的，并且与病毒蛋白表达的急剧降低相关，即使在感染数天的细胞中也是如此。通过单细胞水平的活性蛋白翻译分析，我们表明形成 SG 的感染细胞显示出蛋白质翻译的抑制。总之，我们的数据揭示了一种新的 cbVG 驱动的病毒干扰机制，其中 cbVGs 诱导 PKR 介导的翻译抑制和 SG 形成，导致病毒蛋白表达减少，而不改变整体抗病毒免疫。
