Otter Clayton J, Bracci Nicole, Parenti Nicholas A, Ye Chengjin, Tan Li Hui, Asthana Abhishek, Pfannenstiel Jessica J, Jackson Nathaniel, Fehr Anthony R, Silverman Robert H, Cohen Noam A, Martinez-Sobrido Luis, Weiss Susan R
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Penn Center for Research on Coronaviruses and Other Emerging Pathogens, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
bioRxiv. 2023 Nov 15:2023.11.15.566945. doi: 10.1101/2023.11.15.566945.
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since emerging in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a mutant recombinant SARS-CoV-2 (nsp15) expressing a catalytically inactive nsp15. Infection with SARS-CoV-2 nsp15 led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type (WT) virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15 and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI culture.
自2019年出现以来，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）已导致数百万人死亡。像SARS-CoV-2这样的致死性冠状病毒的先天免疫拮抗作用对于最佳复制和发病机制至关重要。保守的非结构蛋白15（nsp15）核糖核酸内切酶（EndoU）在包括鼠冠状病毒和中东呼吸综合征（MERS）-CoV在内的多种冠状病毒感染期间，限制双链（ds）RNA诱导的途径的激活，包括干扰素（IFN）信号传导、蛋白激酶R（PKR）以及寡腺苷酸合成酶/核糖核酸酶L（OAS/RNase L）。为了确定nsp15在SARS-CoV-2感染过程中的功能，我们构建了一种表达催化失活nsp15的突变重组SARS-CoV-2（nsp15）。与野生型（WT）病毒相比，感染SARS-CoV-2 nsp15导致肺源性上皮细胞系和原代鼻上皮气液界面（ALI）培养物中IFN信号传导和PKR途径的激活增加，以及ALI培养物中复制的显著减弱。当用Janus激活激酶（JAK）抑制剂鲁索替尼抑制IFN信号传导时，这种复制缺陷得到挽救。最后，为了在最小（MERS-CoV）或中度（SARS-CoV-2）先天免疫诱导的背景下评估nsp15的功能，我们比较了SARS-CoV-2 nsp15与先前描述的MERS-CoV nsp15突变体的感染情况。在Calu3细胞和鼻ALI培养物中，nsp15的失活对MERS-CoV复制的影响比对SARS-CoV-2的影响更显著，这表明SARS-CoV-2能够更好地耐受先天免疫反应。综上所述，SARS-CoV-2 nsp15是dsRNA诱导的先天免疫反应的有效抑制剂，其对IFN信号传导的拮抗作用对于原代鼻ALI培养物中的最佳病毒复制是必要的。
