key: cord-0755087-enw4tq2y authors: Rohaim, Mohammed A.; El Naggar, Rania F.; Clayton, Emily; Munir, Muhammad title: Structural and functional insights into non-structural proteins of coronaviruses date: 2020-11-23 journal: Microb Pathog DOI: 10.1016/j.micpath.2020.104641 sha: 3f978056ea514209df30b6564a06cd4795f685a6 doc_id: 755087 cord_uid: enw4tq2y Coronaviruses (CoVs) are causing a number of human and animal diseases because of their zoonotic nature such as Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). These viruses can infect respiratory, gastrointestinal, hepatic and central nervous systems of human, livestock, birds, bat, mouse, and many wild animals. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerging respiratory virus and is causing CoVID-19 with high morbidity and considerable mortality. All CoVs belong to the order Nidovirales, family Coronaviridae, are enveloped positive-sense RNA viruses, characterised by club-like spikes on their surfaces and large RNA genome with a distinctive replication strategy. Coronavirus have the largest RNA genomes (∼26–32 kilobases) and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. Non-structural proteins (nsp) 7–16 are cleaved from two large replicase polyproteins and guide the replication and processing of coronavirus RNA. Coronavirus replicase has more or less universal activities, such as RNA polymerase (nsp 12) and helicase (nsp 13), as well as a variety of unusual or even special mRNA capping (nsp 14, nsp 16) and fidelity regulation (nsp 14) domains. Besides that, several smaller subunits (nsp 7– nsp 10) serve as essential cofactors for these enzymes and contribute to the emerging “nsp interactome.” In spite of the significant progress in studying coronaviruses structural and functional properties, there is an urgent need to understand the coronaviruses evolutionary success that will be helpful to develop enhanced control strategies. Therefore, it is crucial to understand the structure, function, and interactions of coronaviruses RNA synthesizing machinery and their replication strategies. Recent studies have shown that the biochemical characterization of SARS-CoV-2's deubiquitinating and deISGylating behaviours are closer to that of its counterpart in MERS-CoV 154 than that of SARS-CoV. SARS-CoV-2 papain-like protease (PLpro) deISGylating activity 155 appeared to be the most dominant of its diverse proteolytic functions and appeared to be species-156 specific [34] . Additionally, in host cells, nsp3 itself is changed, namely by N-glycosylation of the 157 domain 3Ecto. Nsp3 may also interact with host proteins (such as RCHY1) to promote survival 158 of viruses. Nsp3 was also identified as the largest non-structural protein of CoVs based on a high 159 rate of positively selected mutation sites as the major selective target for driving evolution in Speculating why coronaviruses retain many essential functions in one protein is interesting, 166 while nsp3 protein shows high-rate genetic diversity during CoV evolution. Ultimately, 167 increased research into the structure and function of nsp3 is required to get a more complete 168 understanding of this protein. Initially, the 200-amino-acid-long nsp8 subunit took centre stage due to two reports, the first 225 describing a fascinating hexadecameric structure consisting of eight copies of each of nsp7 and 226 nsp8 [63], and the second revealing a nsp8-specific "secondary" RNA polymerase activity [66] 227 J o u r n a l P r e -p r o o f involved in the CoV RNA synthesis process. Although the structures of feline coronavirus (FCoV; alphacoronavirus 1) nsp7 and nsp8 were found to mimic their SARS-CoV (betacoronavirus) counterparts, two copies of nsp7 and one copy of nsp8 forming a heterotrimer 230 were found to be assembled into a very different higher-order complex [67] . SARS-CoV nsp8 231 was found to adopt two different conformations inside the nsp7-nsp8 hexadecamer. The 232 phylogenetic relationship and similarity percentage of SARS-CoV-2 in relation with other 233 human coronaviruses is shown in Fig. 1a and 1b, respectively. These have been named "golf 234 club" and "bent shaft golf club" [63], with the golf club's globular head considered a new fold. Biochemistry and reverse genetics studies pointed to an important role in RNA synthesis for 236 SARS-CoV nsp8 residues K58, P183, and R190 (Fig. 1c) , replacing which was lethal to SARS- CoV whereas P183 and R190 residues were presumed to be involved in interactions with nsp12, The nsp10 subunit protein (139 residues; SARS-CoV) is one of the most conserved CoV 259 proteins and is believed to serve as an essential multifunctional replication factor. Nsp10 was 260 shown to be dimerized, as well as interact with nsp1, nsp7, nsp14, and nsp16 using yeast two- the innate immune responses, but there was no direct evidence to suggest that in case of viral infection it can counteracts the innate immunity [116] . 3D crystal structure of the nsp15 of 382 SARS-CoV-2 (PDB ID: 6VWW) and the amino acid sequence alignment for nsp8 of SARS- CoV-2 compared to other human coronaviruses (HCoVs) is shown in Fig. 4d and 4c, 384 respectively. Nsp15 can act as a "gatekeeper" for sequestration of viral dsRNA within complex Fig. 5c and 5d , respectively. There is no doubt that further characterization of the "nsp interactome" within the CoV-infected 439 cell will provide more clues about how specific functions are switched on and off or modulated. Understanding these mechanisms will not only highlight their critical roles in the virus 441 replication cycle but may also exposed some key druggable targets to propose novel therapeutics. Intracellular 693 localization and protein interactions of the gene 1 protein p28 during mouse hepatitis virus 694 replication Coronavirus NSP10, a critical co-factor for activation of multiple replicative enzymes The 700 non-structural protein NSP10 of mouse hepatitis virus binds zinc ions and nucleic acids RNA 3'-end mismatch 703 excision by the severe acute respiratory syndrome coronavirus non-structural protein 704 NSP10/NSP14 exoribonuclease complex RNA recognition and 706 cleavage by the SARS coronavirus endoribonuclease Discovery of an essential 710 nucleotidylating activity associated with a newly delineated conserved domain in the RNA 711 polymerase-containing protein of all nidoviruses Coronavirus genome: 713 prediction of putative functional newdomains in the non-structural polyprotein by comparative 714 amino acid sequence analysis Synthesis and biological evaluation of nucleoside analogues having 6-chloropurine as anti-717 SARS-CoV agents The broad spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen Poliovirus pathogenesis in a new poliovirus 722 receptor transgenic mouse model: age dependent paralysis and a mucosal route of infection Coronaviruses-drug discovery and 725 therapeutic options Discovery of an RNA virus 3'-5'exoribonuclease that is critically involved in 772 coronavirus RNA synthesis Functional screen reveals 774 SARS coronavirus nonstructural protein NSP14 as a novel cap N7 methyltransferase Characterization of the guanine-N7 methyltransferase activity of coronavirus NSP14 on 778 nucleotide GTP Structural basis and functional analysis of the SARS coronavirus NSP14-NSP10 complex Potent and selective inhibition of SARS coronavirus 784 replication by aurintricarboxylic acid Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap 787 guanine-N7-methyltransferase The viralRNA capping machinery as a target 789 for antiviral drugs Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits 792 apoptosis in macrophages The coronavirus endoribonuclease NSP15 794 interacts with retinoblastoma tumor suppressor protein Crystal structure and mechanistic determinants of SARS 797 coronavirus non-structural protein 15 define an endoribonuclease family New antiviral target revealed by the hexameric structure of mouse hepatitis 801 virus nonstructural protein NSP15 Severe acute respiratory 806 syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate 807 antagonism of IRF3 and NF-κB signaling Early endonuclease-mediated evasion of RNA 812 sensing ensures efficient coronavirus replication mRNA cap-1 methyltransferase in the 814 SARS genome Coronavirus nonstructural protein 16 is a cap-0 binding enzyme 817 possessing (nucleoside-2'O)-methyltransferase activity Pathogenic influenza viruses and 822 coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene 823 responses Attenuation and restoration of severe acute respiratory syndrome coronavirus 826 mutant lacking 2′-o-methyltransferase activity Coronavirus NSP10/ NSP16 methyltransferase can be targeted by NSP10-derived 848 peptide in vitro and in vivo to reduce replication and pathogenesis Short peptides derived from the interaction domain of SARS coronavirus nonstructural 852 protein NSP10 can suppress the 2'-O-methyltransferase activity of NSP10/NSP16 complex Construction of a severe acute 856 respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus 857 RNA synthesis Ribose 859 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA 860 dependent on the RNA sensor Mda5 • Coronavirus disease 2019 (COVID-19) is causing devastating impacts around the world with over a million deaths and 50 million infections ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:J o u r n a l P r e -p r o o f