key: cord-0921480-cnpwirev authors: Lang, Yuekun; Chen, Ke; Li, Zhong; Li, Hongmin title: The nucleocapsid protein of zoonotic betacoronaviruses is an attractive target for antiviral drug discovery date: 2020-11-12 journal: Life Sci DOI: 10.1016/j.lfs.2020.118754 sha: 1dc178653fd2a5c22e2cf1f726cee8de96ad2a0b doc_id: 921480 cord_uid: cnpwirev Betacoronaviruses are in one genera of coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), etc. These viruses threaten public health and cause dramatic economic losses. The nucleocapsid (N) protein is a structural protein of betacoronaviruses with multiple functions such as forming viral capsids with viral RNA, interacting with viral membrane protein to form the virus core with RNA, binding to several cellular kinases for signal transductions, etc. In this review, we highlighted the potential of the N protein as a suitable antiviral target from different perspectives, including structure, functions, and antiviral strategies for combatting betacoronaviruses. There are four genera of coronaviruses (CoVs) in the subfamily Orthocoronavirinae of Coronaviridae family: alphacoronaviruses (α-CoVs), betacoronaviruses (β-CoVs), gammacoronaviruses (γ-CoVs), and deltacoronaviruses (δ-CoVs) 1 . CoVs have been identified in different mammals and fowl such as dogs, cats, horses, bats, cattle, swine, mice, whales, monkeys, ferrets, camels, turkeys, and chickens 2 9 . In this review, we will mainly focus on the antiviral aspects of the N protein in zoonotic β-CoVs. All three domains are related to RNA binding 18, 19 . Because of the characteristics of the N protein, such as poor stability and dynamic behavior, no crystal structures were solved for fulllength N proteins of coronaviruses. In solution, the full-length SARS-CoV N protein predominantly exists as a CTD dimer that is considered to be the basic building blocks of the nucleocapsid ( Figure. 1D) 20, 21, 22 . A structural model for a di-domain (DD) has been proposed by fitting small angle X-ray scattering data for the SARS-CoV N protein 23 . The CTD dimer forms as a core with NTDs branching out as two arms connecting to the core via CLR (Figure. Although NTDs are divergent in both sequence and length within Coronaviridae 24 , they are relatively conserved within Betacoronavirus genera. As shown in Figure 2 , NTD has been mapped for HCoV-OC43 (aa 25 , SARS-CoV-2 (aa 46-174) 26 , SARS-CoV (aa 45-181) 27 , and MERS-CoV (aa 39-165) 28 . NTD within zoonotic β-CoVs displays a right-handed (loops)-(β-sheet core)-(loops) sandwiched structure which is conserved among all NTD in CoVs ( Figure 2 ) 26, 29 . The hand-shaped NTD is represented by basic fingers, a hydrophobic basic palm, and an acidic wrist ( Figure. 1E) 26, 30 . The hand-shaped NTD residues in the middle part are more conserved than residues located in the basic fingers and acidic wrist 26 . Based on the crystal The NTD and CTD are linked by CLR, which is rich in serine and arginine residues. The CLR also contains abundant of phosphorylation sites which facilitate the N protein to become involved in cell signaling 34, 35, 36, 37, 38 . The flexibility of CLR facilitates its direct interactions between the N protein and RNAs 27, 23, 39, 40, 41 . However, because there is no structural information for the CLR, and protein phosphorylation is reversible, opposing hypotheses on the function of CLR in N protein oligomerization have been proposed based on different facts. Because phosphorylation can stabilize the N protein by reducing the total positive charge, hyperphosphorylation of CLR could enhance and regulate oligomerization of DD 42 . In another study, it was reported that oligomerization might be impaired when the CLR is phosphorylated 43, 44 . These results suggest that phosphorylation may act as a key to lock or unlock N protein oligomerization. Similar to that of NTD, sequence and length of the CTD are relatively conserved within the β-CoV genera, indicating similar structural and functional roles for the CTD 45 . The monomer of the SARS-CoV N protein is unstable because the CTD folds into an extended conformation with a topology of α1-α2-α3-α4-α5-α6-β1-β2-α7-α8, leading to a large cavity in its center 45, 46 . The CTD can be stabilized through domain-swapped dimerization by inserting a βhairpin of one subunit into the center cavity of the opposite subunit 21, 22, 45, 46, 47, 48, . Exploration characterized by a continuous positively charged surface 46 . Through electrostatic interactions between the continuous positively charged surface of the CTD supercomplex and negatively charged RNA strands, the viral RNA strands can bind and wrap around the CTD supercomplex by non-specific charge interactions ( Figure. 1G, H) 46, 49 . Similar to human CoV 229E (hCoV-229E), all zoonotic β-CoV NPs have C-terminal tail peptides. In hCoV-229E, the disordered Cterminal tail is responsible for dimer-dimer association. It has been reported that peptide N377-389 from the C-terminus of hCoV-229E has an inhibitory effect on viral titer of HCoV-229E 47 . Understanding the mechanism of how the C-terminal tail peptide in the β-CoV N protein is involved in oligomerization may shed light on identifying antiviral targets for drug discovery to combat β-CoVs by means of disrupting the N protein self-association. The primary function of the β-CoV N protein is to package the viral genomic RNA into nucleocapsids. This structure not only protects the genome, but also further guarantees that the replication and transmission can be done in a timely and reliable manner. Therefore, a correct and stable structure is important for nucleocapsid function. The β-CoV N protein uses its dimer form as a basic building block for nucleocapsid formation 30 . Based on the crystal structure of SARS-CoV CTD, the dimerization core has been identified at aa 281-365 46 . The N protein, M protein, and gRNA form the internal spherical/icosahedral core of CoVs 50, 51 . Although the M protein can form virus-like particle (VLP) alone via self-assembly, Since the 1960s, antivirals targeting many different viral diseases have been identified based on different mechanisms 78 However, we need to carefully consider putting these drugs into human clinical trials. In addition to potential side effects, antiviral drugs may worsen the situation if drug-resistant strains emerge. Journal Pre-proof Also, unlike most antibiotics, specific antivirals are designed to target specific viral proteins that may be involved in normal human functions instead of simply inactivating the pathogen 95 . 96 . In this review, we focus on antiviral strategies targeting the viral N protein. Since the N protein of β-CoVs is a multifunctional structural protein with conserved In addition, because only high-biosafety-level labs can handle the virulent strains of zoonotic β-CoVs, hindering the speed of antiviral discovery, it is crucial to find suitable models to study the antivirals in lower-biosafety-level labs to initiate the primary screening of antiviral candidates. VLPs without infectious properties should be functional and safe models for this purpose 50 . For example, based on N protein functions, we know it mainly serves as a structural protein. So, if the conformation of the N protein changes, VLPs might not be able to process successfully 101, 102 . Thus, we can use VLPs to screen antiviral candidates at the cell base level. Currently, we have an urgent unmet medical need for antiviral drugs with different mechanisms to combat β-CoV pandemics or epidemics. 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HL was partially supported by NIH grants AI131669, AI133219, AI140491, AI134568, AI140406, AI141178, and AI140726. The authors declare no competing financial interest.