SARS-CoV-2 is an enveloped, positive-sense RNA virus of public health concern due to its zoonotic potential and its role as the causative agent of the COVID-19 pandemic. The Nsp15 of SARS-CoV-2 is a homohexamer consisting of six protomers. Nsp15 cleaves the 3’ end of uridine in dsRNA intermediates to help the virus to escape from the dsRNA sensors. Determining the impact of mutations on the structure and function of Nsp15 could yield insights into developing inhibitors that reduce the ability of the virus to circumvent host immunity and to understand the mechanism of mutated Nsp15. This study aims to understand how a natural T112I mutation in SARS-CoV-2 Nsp15 helps the virus to evade the immune system. The T112I mutation is characterized by the significant substitution of a polar residue (threonine) with a hydrophobic residue (isoleucine). The T112 residue may also be critical for Nsp15 activity because it is in the RNA-binding domain of Nsp15. Therefore, we hypothesized that the T112I mutation is favorable for RNA binding through conformational changes, which may lead to enzyme hyperactivity. The goal of this research is to determine the high-resolution structure of the apo Nsp15 T112I mutant using X-ray crystallography, to identify the structural changes responsible for its hyperactivity. In addition, as a foundational step toward solving the double-stranded RNA-bound structure, the uridine monophosphate (UMP)-bound structure of the Nsp15 T112I mutant will first be determined to study the substrate binding characteristics of this hyperactive enzyme. Moreover, functional analysis will be conducted using nuclease assays to characterize and quantify the enzymatic activity of the Nsp15 T112I protein. These findings will provide insights into the cleavage mechanism of mutant Nsp15, facilitating the development of therapeutic interventions to attenuate SARS-CoV-2 and its emerging variants.
Kankanamalage et al. (Sun,) studied this question.