Sonowal Manashi, Ketawala Gihan, Nagaratnam Nirupa, Logeswaran Dhenugen, Basu Shibom, de Sanctis Daniele, Orlans Julien, Rose Samuel L, Jernigan Rebecca J, Hu Hao, Aguilar Jose Domingo Meza, Ranaweera Madurangi E, Zacks Michele A, Chen Julian J-L, Hansen Debra T, Schrag Lynn G, Fromme Raimund, Botha Sabine, Fromme Petra
Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA.
Protein Sci. 2025 Jun;34(6):e70115. doi: 10.1002/pro.70115.
SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has undergone continuous evolution, leading to the emergence of variants with altered transmissibility and immune evasion. For the non-structural proteins (Nsps) of SARS-CoV-2, there are limited structural analyses of their naturally occurring mutations. Here, we identified four non-synonymous single-nucleotide polymorphisms (nsSNPs) in the Epsilon lineage of SARS-CoV-2 within Nsp15, an endoribonuclease critical for immune evasion. Of these Epsilon nsSNPs, E266Q is in the catalytic domain. This study investigates the effects of this on enzymatic activity, structural stability, and oligomeric assembly by serial crystallography. By solving the structure of the Nsp15 hexamer at room temperature of both Nsp15-E266Q and WT in the P2 space group to 3 Å, we observed asymmetric motions within its trimer subunits, a feature not visible in previously reported higher-symmetry space groups. These asymmetric motions resemble substrate-induced conformational changes reported in RNA-bound Nsp15 structures, suggesting functional relevance. Biochemical analyses further reveal that Nsp15-E266Q exhibited significantly higher enzymatic activity and thermal stability compared to the wild-type protein. These findings highlight how mutations in Nsp15 contribute to viral replication and immune evasion, offering insights into the molecular mechanisms underlying SARS-CoV-2 variant evolution and potential therapeutic strategies.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是导致新冠疫情的病毒，它不断进化，导致出现了传播性和免疫逃逸能力改变的变种。对于SARS-CoV-2的非结构蛋白（Nsps），对其天然发生的突变进行的结构分析有限。在这里，我们在SARS-CoV-2的Epsilon谱系的Nsp15中鉴定出四个非同义单核苷酸多态性（nsSNPs），Nsp15是一种对免疫逃逸至关重要的核糖核酸内切酶。在这些Epsilon nsSNPs中，E266Q位于催化结构域。本研究通过系列晶体学研究了这一突变对酶活性、结构稳定性和寡聚体组装的影响。通过在P2空间群中将Nsp15-E266Q和野生型Nsp15六聚体在室温下的结构解析到3 Å，我们观察到其三聚体亚基内的不对称运动，这一特征在先前报道的更高对称性空间群中不可见。这些不对称运动类似于在RNA结合的Nsp15结构中报道的底物诱导的构象变化，表明其具有功能相关性。生化分析进一步表明，与野生型蛋白相比，Nsp15-E266Q表现出显著更高的酶活性和热稳定性。这些发现突出了Nsp15中的突变如何促进病毒复制和免疫逃逸，为SARS-CoV-2变种进化的分子机制和潜在治疗策略提供了见解。
