Walter Marius, Chen Irene P, Vallejo-Gracia Albert, Kim Ik-Jung, Bielska Olga, Lam Victor L, Hayashi Jennifer M, Cruz Andrew, Shah Samah, Gross John D, Krogan Nevan J, Schilling Birgit, Ott Melanie, Verdin Eric
Buck Institute for Research on Aging, Novato, CA, United States.
Gladstone Institutes, San Francisco, CA, United States.
bioRxiv. 2022 Jan 5:2022.01.04.474979. doi: 10.1101/2022.01.04.474979.
SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）非结构蛋白Nsp14是病毒复制所必需的一种高度保守的酶。Nsp14与非结构蛋白Nsp10形成稳定复合物，并具有外切核糖核酸酶和N7-甲基转移酶活性。蛋白质相互作用组研究确定人类沉默调节蛋白5（SIRT5）是Nsp14的一个假定结合伴侣。SIRT5是一种对细胞代谢至关重要的NAD依赖性蛋白质去酰基酶，可从赖氨酸残基上去除琥珀酰基和丙二酰基。在此，我们研究了这种相互作用的性质以及SIRT5在SARS-CoV-2感染过程中的作用。我们发现SIRT5与Nsp14稳定相互作用，但与Nsp10不相互作用，这表明SIRT5和Nsp10是不同复合物的组成部分。我们发现SIRT5催化结构域对于与Nsp14的相互作用是必需的，但Nsp14似乎不会被SIRT5直接去酰化。此外，在细胞培养实验中，敲除SIRT5或用特定的SIRT5抑制剂处理可降低SARS-CoV-2病毒水平。敲除SIRT5的细胞表达更高水平的基础先天免疫标志物，并产生更强的抗病毒反应。我们的结果表明，SIRT5是有效病毒复制所必需的一种病毒促进因子，这为治疗干预开辟了新途径。
