Milan Unit, National Research Council-Institute for Genetic and Biomedical Research, 20138 Milan, Italy.
Human Genome and Biomedical Technologies Unit, IRCCS Humanitas Research Hospital, 20089 Milan, Italy.
Int J Mol Sci. 2023 Jan 30;24(3):2634. doi: 10.3390/ijms24032634.
Upon infection, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is predicted to interact with diverse cellular functions, such as the nonsense-mediated decay (NMD) pathway, as suggested by the identification of the core NMD factor upframeshift-1 (UPF1) in the SARS-CoV-2 interactome, and the retrograde transport from the Golgi to the endoplasmic reticulum (ER) through the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where coronavirus assembly occurs. Here, we investigated the expression and localization of the neuroblastoma-amplified sequence (NBAS) protein, a UPF1 partner for the NMD at the ER, participating also in retrograde transport, and of its functional partners, at early time points after SARS-CoV-2 infection of the human lung epithelial cell line Calu3. We found a significant decrease of DExH-Box Helicase 34 (, suppressor with morphogenetic effect on genitalia 5 (, and expression at 6 h post-infection, followed by a significant increase of these genes and also and at 9 h post-infection. Conversely, and other genes coding for NMD factors were not modulated. Known NMD substrates related to cell stress (Growth Arrest Specific 5, transducin beta-like 2, ; and DNA damage-inducible transcript 3, ) were increased in infected cells, possibly as a result of alterations in the NMD pathway and of a direct effect of the infection. We also found that the expression of unconventional SNARE in the ER 1, (p31) and Zeste White 10 homolog, , partners of NBAS in the retrograde transport function, significantly increased over time in infected cells. Co-localization of NBAS and UPF1 proteins did not change within 24 h of infection nor did it differ in infected versus non-infected cells at 1 and 24 h after infection; similarly, the co-localization of NBAS and p31 proteins was not altered by infection in this short time frame. Finally, both NBAS and UPF1 were found to co-localize with SARS-CoV-2 S and N proteins. Overall, these data are preliminary evidence of an interaction between NBAS and NBAS-related functions and SARS-CoV-2 in infected cells, deserving further investigation.
在感染后，严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）被预测会与多种细胞功能相互作用，例如无意义介导的衰变（NMD）途径，这是通过鉴定 SARS-CoV-2 相互作用组中的核心 NMD 因子 upframeshift-1（UPF1）以及通过内质网-高尔基体中间区（ERGIC）从高尔基体逆行运输到内质网（ER）而得出的，冠状病毒的组装就是在此处发生的。在这里，我们研究了神经母细胞瘤扩增序列（NBAS）蛋白的表达和定位，该蛋白是内质网中 NMD 的 UPF1 伴侣，也参与逆行运输，以及其功能伴侣在 SARS-CoV-2 感染人肺上皮细胞系 Calu3 后的早期时间点的表达。我们发现，在感染后 6 小时，DEXH-Box Helicase 34（DDX34）、生殖器形态发生效应物 5（SMG5）和 表达显著下降，随后这些基因以及 和 表达显著增加，而 9 小时后也是如此。相反，其他 NMD 因子基因没有被调节。已知与细胞应激相关的 NMD 底物（生长停滞特异性 5（GAS5）、转导素β样 2（TBL2）和 DNA 损伤诱导转录物 3（DDIT3））在感染细胞中增加，可能是由于 NMD 途径的改变和感染的直接影响。我们还发现，在内质网中非常规 SNARE1、 （p31）和 Zeste White 10 同源物 （ZW10）的表达显著增加，NBAS 在逆行运输功能中的伴侣，随着时间的推移，在感染细胞中显著增加。在感染后 24 小时内，NBAS 和 UPF1 蛋白的共定位没有改变，在感染和未感染细胞中，感染后 1 小时和 24 小时的共定位也没有差异；同样，在这个短时间内，感染也没有改变 NBAS 和 p31 蛋白的共定位。最后，NBAS 和 UPF1 都被发现与 SARS-CoV-2 S 和 N 蛋白共定位。总的来说，这些数据初步证明了 NBAS 及其相关功能与感染细胞中的 SARS-CoV-2 之间存在相互作用，值得进一步研究。
