key: cord-0704929-e4c26nn9 authors: Angeletti, Silvia; Benvenuto, Domenico; Bianchi, Martina; Giovanetti, Marta; Pascarella, Stefano; Ciccozzi, Massimo title: COVID‐2019: The role of the nsp2 and nsp3 in its pathogenesis date: 2020-02-28 journal: J Med Virol DOI: 10.1002/jmv.25719 sha: ae37dcd492c6ed8068cbd22a740aef89dd5d8f46 doc_id: 704929 cord_uid: e4c26nn9 Last December 2019, a new virus, named novel Coronavirus (COVID‐2019) causing many cases of severe pneumonia was reported in Wuhan, China. The virus knowledge is limited and especially about COVID‐2019 pathogenesis. The Open Reading Frame 1ab (ORF1ab) of COVID‐2019 has been analyzed to evidence the presence of mutation caused by selective pressure on the virus. For selective pressure analysis fast‐unconstrained Bayesian approximation (FUBAR) was used. Homology modelling has been performed by SwissModel and HHPred servers. The presence of transmembrane helical segments in Coronavirus ORF1ab non structural protein 2 (nsp2) and nsp3 was tested by TMHMM, MEMSAT, and MEMPACK tools. Three‐dimensional structures have been analyzed and displayed using PyMOL. FUBAR analysis revealed the presence of potential sites under positive selective pressure (P < .05). Position 723 in the COVID‐2019 has a serine instead a glycine residue, while at aminoacidic position 1010 a proline instead an isoleucine. Significant (P < .05) pervasive negative selection in 2416 sites (55%) was found. The positive selective pressure could account for some clinical features of this virus compared with severe acute respiratory syndrome (SARS) and Bat SARS‐like CoV. The stabilizing mutation falling in the endosome‐associated‐protein‐like domain of the nsp2 protein could account for COVID‐2019 high ability of contagious, while the destabilizing mutation in nsp3 proteins could suggest a potential mechanism differentiating COVID‐2019 from SARS. These data could be helpful for further investigation aimed to identify potential therapeutic targets or vaccine strategy, especially in the actual moment when the epidemic is ongoing and the scientific community is trying to enrich knowledge about this new viral pathogen. protein could account for COVID-2019 high ability of contagious, while the destabilizing mutation in nsp3 proteins could suggest a potential mechanism differentiating COVID-2019 from SARS. These data could be helpful for further investigation aimed to identify potential therapeutic targets or vaccine strategy, especially in the actual moment when the epidemic is ongoing and the scientific community is trying to enrich knowledge about this new viral pathogen. Knowledge about COVID-2019 is still incomplete, many questions have raised and many answers are needed first of all regarding its pathogenicity, its ability to change, how many people will get sick from each infected person, the so-called R0 and when infection will be preventable or treatable. 3 In the last period where many researchers are intensively studying the mechanism of COVID-2019 replication, pathogenicity, and therapeutic strategies, the present study has been Table 1 ). The pairwise percentage of similarity has been calculated using Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi); duplicated sequences have been removed from the dataset. The 25 sequences have been aligned using a multiple sequence alignment multiple alignment using fast fourier transform online tool 4 and manually edited using Bioedit program v7.0.5. 5 The selective pressure analysis was focused on the polyprotein ORF1ab because it differs from the most similar bat Coronavirus (QHR63299) for only 103 amino acid residues, 64 of them are conservative changes. In particular, non structural protein 2 (nsp2) differs from bat Coronavirus for 11 residues while nsp3 for 64 residues of which 44 are conservative changes. Adaptive Evolution Server (http://www.datamonkey.org/) was used to find eventual sites under of positive or negative selection pressure. At this purpose the following tests has been used: fastunconstrained Bayesian approximation (FUBAR). 6 These tests allowed to infer the site-specific pervasive selection, the episodic diversifying selection across the region of interest and to identify episodic selection at individual sites. 7 Statistically significant positive or negative selection was based on P < .05. Homology modelling has been attempted with SwissModel 8 and HHPred 9 servers. Models for ORF1ab nsp2 and nsp3 proteins available at the I-Tasser web site (corresponding to codes QHD43415_2 and QHD43415_3) 10 have been considered. PDB Proteins structurally close to the target have been evaluated using the TM-score 11 while the RAMPAGE 12 online tool has been used to assess the folding quality of the model. The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health-The latest 2019 novel coronavirus outbreak in Wuhan, China The 2019-new coronavirus epidemic: evidence for virus evolution Overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses SARS-CoV, MERS-CoV, and 2019-nCoV MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization BioEdit A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT FUBAR: a fast, unconstrained bayesian approximation for inferring selection Detecting individual sites subject to episodic diversifying selection SWISS-MODEL: homology modelling of protein structures and complexes A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core The I-TASSER suite: Protein structure and function prediction Scoring function for automated assessment of protein structure template quality Structure validation by Calpha geometry: phi, psi, and Cbeta deviation Evaluation of methods for the prediction of membrane spanning regions Transmembrane protein topology prediction using support vector machines The MEMPACK alpha-helical transmembrane protein structure prediction server The {PyMOL} Molecular Graphics System. Version 1.80 LLC Structural basis of severe acute respiratory syndrome coronavirus ADP-ribose-1''-phosphate dephosphorylation by a conserved domain of nsP3. Structure Clinical features of patients infected with 2019 novel coronavirus in Wuhan China novel coronavirus investigating and research team. A novel coronavirus from patients with pneumonia in China Emerging understandings of 2019-nCoV The first two cases of 2019-nCoV in Italy: where they come from Three emerging coronaviruses in two decades COVID-2019: The role of the nsp2 and nsp3 in its pathogenesis