1. Introduction
The SARS-CoV-2 genome includes two large open reading frames (ORFs/ORF1ab) that occupy more than two-thirds of its length[1]. ORF1ab is proteolytically processed by two virally encoded proteinases (PLpro and Mpro) to produce 16 non-structural proteins (Nsp1 to Nsp16)[2,3]. Nsp1 is encoded by the gene closest to the 5′-end of the genome, being also known as leader protein[4,5]. Nsp1 is believed to have identical physical features and biological activity among β-coronaviruses, although being poorly conserved [5,6]. Overall, Nsp1 has 180 amino acids (aa) forming two unstructured domains and one domain working as an interconnecting region between the two unstructured domains[7]. The crystal structure of SARS-CoV-2 Nsp1 revealed a six-stranded conformation [8], with a C-terminal region holding an intrinsically disordered domain (residues 130-180) in comparison with its N-terminal region[9,10]. Based on previous studies, the interaction between the helical hairpin at the C-terminal domain of Nsp1 and the host 40S subunit of ribosome prohibits the entry of mRNA into the host ribosome [9]. Therefore, Nsp1 leads to translational shutoff of proteins from host mRNAs by binding the 40S ribosome of the host cell [9,11,12]. The C-terminal domain of Nsp1 has two helices [11]that are inserted into the entrance area of the ribosomal mRNA channel, probably contributing to the translational blockage[9,11]. In fact, Nsp1 leads to an endonucleolytic cleavage near the 5′ UTR of host mRNA (capped mRNA) when binding to ribosome [13]. Moreover, Nsp1 inhibits all cellular antiviral defense mechanisms such as Type I interferon (IFN‐I) response, which leads to the shutoff of the response of the innate immune system [11,14]. Examination of the physical properties of the SARS-CoV-2 Nsp1 has shown that has a positive surface charge [15]. Despite the existence of a structural model of the Nsp1 [9,16], few studied have been conducted on the dynamic aspects of this protein and how mutations may be change its conformation and function. Here, we used molecular dynamic (MD) and normal mode analysis (NMA) to further investigate the structure of Nsp1. We compared SARS-CoV-2 and SARS-CoV-1 Nsp1 and determined the effect of over 500 mutations on Nsp1 stability. These observations might highlight the relevance of understanding how mutations effect the Nsp1 inhibition of immune response and binding to the ribosome.