New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2

Abstract

Open AccessCCS ChemistryMINI REVIEW1 Jan 2021New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2 Zilong Zhao†, Yaling Wang†, Liping Qiu†, Ting Fu, Yu Yang, Ruizi Peng, Mengyu Guo, Lichun Mao, Chunying Chen, Yuliang Zhao and Weihong Tan Zilong Zhao† Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 , Yaling Wang† CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 , Liping Qiu† Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 , Ting Fu Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 The Cancer Hospital of the University of Chinese Academy of Sciences, Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, Zhejiang 310022 , Yu Yang Institute of Molecular Medicine (IMM), Renji Hospital, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240 , Ruizi Peng Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 , Mengyu Guo CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 , Lichun Mao CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 , Chunying Chen *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 GBA Research Innovation Institute for Nanotechnology, Guangdong 510700 , Yuliang Zhao CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 GBA Research Innovation Institute for Nanotechnology, Guangdong 510700 and Weihong Tan *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 The Cancer Hospital of the University of Chinese Academy of Sciences, Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, Zhejiang 310022 Institute of Molecular Medicine (IMM), Renji Hospital, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240 https://doi.org/10.31635/ccschem.020.202000322 SectionsAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly sweeping across all countries, bringing social and economic hardship to millions. Most countries have implemented early warning measures to detect, isolate, and treat patients infected with SARS-CoV-2. This minireview summarizes some of those steps, in particular, testing methods and drug development in the context of chemical biology, and discusses the molecular basis of COVID-19's virulent transmissibility. Download figure Download PowerPoint Introduction Coronavirus disease 2019 (COVID-19), which emerged in December 2019, is caused by a novel strain of coronavirus (CoV), designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of July 29, 2020, the virus had attributed to more than 16.5 million laboratory-confirmed cases of SARS-CoV-2 infection with 655,112 deaths, sweeping virtually every country worldwide.1 Initial COVID-19 cases presented pneumonia-like symptoms with abnormal lung scanned computed tomography (CT) images. These cases were determined initially to be a "pneumonia of unknown etiology" by clinicians, according to the following criteria: fever (≥38 °C), radiographic evidence of pneumonia, low or normal white cell count or low lymphocyte count, and no symptomatic improvement after treatment with antibiotics for 3–5 days, following standard clinical guidelines.2,3 On January 5, 2020, the causative agent of the "pneumonia of unknown etiology" was identified as a SARS-CoV-2 by deep sequencing and etiological investigations.4 The genome of the novel virus was about 80% identical to that of SARS-CoV, which caused the outbreak of severe acute respiratory syndrome in 2003.5–7 Based on phylogeny, taxonomy, and established practice, the novel virus was designated as SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV).8 The introduction of CoV CoVs belong to the subfamily Coronavirinae in the family of Coronaviridae of the order Nidovirales and are characteristic enveloped, single-stranded, and positive-sense RNA viruses.9 As observed under electron microscopy (EM), CoVs are named based on their pleomorphic or spherical shape (diameter 80–120 nm) and further characterization as bearing club-shaped peplomers on the surface (Figure 1a). CoVs possess the largest genomes (26.4–31.7 kb) in spherical capsid among all known RNA viruses. Based on serological and genetic properties, CoVs are subdivided into four genera: alpha-, beta-, delta-, and gammacoronavirus.10 Their host range includes humans and several other vertebrates, typically causing respiratory, digestive, and nervous system diseases. Up to now, seven human CoVs (hCoVs) have emerged, including hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1, SARS-CoV, Middle-East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2. Among these hCoVs, SARS-CoV-2, MERS-CoV, and SARS-CoV have crossed the species barrier to cause deadly pneumonia in humans in the first 20 years of the 21st century.1,11,12 Figure 1 | (a) Transmission electron microscopy (TEM) of SARS-CoV-2. Reproduced with permission from ref 7. Copyright 2020 China CDC. (b) Schematic of SARS-CoV-2, which is probably a fair representation. Download figure Download PowerPoint The genome and proteins of SARS-CoV-2 The genetic makeup of SARS-CoV-2 comprises ∼29.9 nucleotides, and its genome organization is similar to two representative members of the genus betacoronavirus: SARS-CoV Tor2, a CoV associated with humans, and bat SL-CoVZC45, a CoV associated with bats (Figure 2).13–15 The genome of SARS-CoV-2 is predicted to contain 14 open reading frames (ORFs) encoding 29 proteins. Its gene order is ORF1a/b, spike (S), envelope (E), membrane (M), and nucleocapsid (N) in 5′ to 3′ direction. The 265-nt 5′-terminal and 229-nt 3′-terminal sequences are characterized as betacoronaviruses. The ORF1a/b gene (21,291 nt), about two-thirds of the SARS-CoV-2 genome, encodes two viral polypeptides: PP1a and PP1 ab. These polypeptides are further processed by virus-encoded chymotrypsin-like protease (3CLpro) or main protease (Mpro, also called 3C-like protease), producing 16 predicted nonstructural proteins (NSPs), including nsp1 to nsp16. These NSPs are necessary for replication and transcription. Figure 2 | Genome organization of SARS-CoV-2 and two SARS-CoVs: bat SL-CoVZC45 and SARS-CoV Tor2. Reproduced with permission from ref 13. Copyright 2020 Springer Nature. Download figure Download PowerPoint Additionally, the SARS-CoV-2 comprises structural proteins, with genes located among the ORF genes, ORF10 and ORF11, predicted to encode four main viral proteins: S, E, M, and N (Figure 1b).14 The M is a matrix glycoprotein is the most abundant structural protein, characterized as a triple membrane-spanning protein with a short NH2 terminus outside the virus and a long COOH terminus inside the virion. S glycoprotein is a single-pass type I membrane-anchored protein and constitutes the peplomers, the glycoprotein spikes that stud the viral capsid. N protein is a nucleocapsid that binds and interacts with viral genomic RNA to form the helical nucleocapsid. The E protein is a small membrane envelope protein involved in the assembly of the virus and its budding, playing a role in the viral pathogenesis. A molecular interaction that might determine the formation and composition of the coronaviral membrane exists between the E proteins. The remaining genome part of SARS-CoV-2 produces eight predicted accessory proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9a, and ORF9b), which might be necessary for the viral virulence in vivo, but their actual biological functions are undefined. Since the outbreak of SARS-CoV-2, researchers have rapidly obtained the crystal structures of SARS-CoV-2-related proteins (e.g., S glycoprotein and Mpro). Ensuing research has produced valuable data facilitating our understanding of the infectivity mechanism and human-to-human transmission of SARS-CoV-2, effectively laying the foundation for the development of therapeutics and vaccines. Molecular basis of SARS-CoV-2 transmission SARS-CoV entry into host cells is mediated by the S (spike) glycoprotein which recognizes angiotensin-converting enzyme 2 (ACE2) on host cells via the formation of homotrimers.16–18 S glycoprotein of SARS-CoV consists of two functional subunits: S1 subunit and S2 subunit. The S1 subunit contains the receptor-binding domain (RBD) responsible for specific binding to ACE2 on host cells. The S2 subunit is responsible for virus-cell fusion. SARS-CoV-2 shares several highly homologous patches of sequences in the RBD domain with SARS-CoV; however, four of five key residues, which are reported to be critical for cross-species and human-to-human transmission of SARS-CoV, are mutated in SARS-CoV-2.6 Therefore, to determine whether SARS-CoV-2 infectivity is mediated by ACE2, Zhou et al.16 systematically investigated the entry of SARS-CoV-2 in HeLa cells that expressed or did not express ACE2 receptors from different species, including human, Chinese horseshoe bats, civets, pigs, and mice. The results confirmed that all ACE2 receptors, except mouse ACE2, mediate the entry of SARS-CoV-2 into HeLa cells (Figure 3). They also identified other CoV receptors, such as aminopeptidase (APN) and dipeptidyl peptidase 4 (DPP4), but these were not classified as cellular entry receptors of SARS-CoV-2. The results strongly supported the notion that SARS-CoV-2 infectivity in the host cell is mediated by ACE2. The binding affinity between SARS-CoV-2 and ACE2 was also investigated by different research groups.19–22 These results suggested that the binding affinity between SARS-CoV-2 and hACE2 is better than that of SARS-CoV and hACE2, although the exact dissociation constant (Kd) values vary depending on the specific experimental operations (Table 1). Figure 3 | Analysis of SARS-CoV-2 infectivity in HeLa cells that expressed or did not express ACE2 from various species. hACE2, human ACE2; bACE2, ACE2 of Rhinolophus sinicus (bat); cACE2, civet ACE2; sACE2, swine ACE2 (pig); mACE2, mouse ACE2. All ACE2 receptors with S tags can be detected using mouse anti-S tag monoclonal antibody. Green, ACE2; red, viral protein (N); blue, 4',6-diamidino-2-phenylindole (DAPI) (nuclei). Scale bars, 10 μm. Reproduced with permission from ref 16. Copyright 2020 Springer Nature. Download figure Download PowerPoint Table 1 | Analysis of Binding Affinities Between RBD and hACE2 SARS-CoV-2 SARS-CoV Measuring Method References Kd (nM) Kon (M−1 S−1) Koff (S−1) Kd (nM) Kon (M−1 S−1) Koff (S−1) 1.2 ± 0.1 2.3 ± 1.4 × 105 1.7 ± 0.8 × 10−4 5.0 ± 0.1 1.7 ± 0.7 × 105 8.7 ± 5.1 × 10−4 Biolayer interferometry 20 44.2 1.75 × 105 7.75 × 10−3 185 2.01 × 105 3.70 × 10−2 Surface plasmon resonance 19 4.674 1.400 × 106 6.544 × 10−3 31.59 1.367 × 106 4.317 × 10−2 21 94.6 ± 6.5 4.0 ± 0.2e4 3.8 ± 0.1e-3 408.7 ± 11.1 2.9 ± 0.2e5 1.9 ± 0.4e-3 22 To elucidate the molecular basis for SARS-CoV-2 infectivity and virulent human-to-human transmission, many research groups have committed to discovering the crystal structure of the interacting interface between SARS-CoV-2 RBD and ACE2.19–24 These findings could shed light on the molecular mechanism of virulent human-to-human transmission, facilitate the understanding the viral infectivity, and provide targets for drug development. For example, Shang et al.19 designed a SARS-CoV-2 chimeric RBD, consisting of a core from SARS-CoV RBD as the crystallization scaffold, and the receptor-binding motifs (RBMs), containing most of the contacting residues of virus for ACE2 binding from SARS-CoV-2 as the functional unit. To enhance the crystallization of the complex, a short loop from SARS-CoV RBD maintaining a strong salt bridge between Arg426 from the RBD and Glu from hACE2 was retained. X-ray diffraction revealed that the overall structure of the chimeric RBD/hACE2 complex and that of the SARS-CoV RBD/hACE2 complex bear subtle similarities in conformational changes, despite the presence of mutations in some residues (Figure 4a). Both chimeric RBD and SARS-CoV RBD cradle hACE2 N-terminal helix by forming a gently concave surface with a ridge on one side. One structural difference between the RBMs of chimeric RBD and SARS-CoV RBD involves the conformation of loops in the hACE2-binding ridge. As shown in Figures 4b and 4c, the ridge loop of SARS-CoV RBD contains a three-residue motif, proline-proline-alanine, between the disulfide-bond-forming cysteines, allowing the loop to take a sharp turn. The ridge loop of chimeric RBD contains a four-residue motif, glycine-valine/glutamine-glutamate/threonine-glycine, allowing the loop to take a different conformation. The structural difference, together with an additional main-chain hydrogen bond between Asn487 and Ala475 in SARS-CoV-2 RBM, makes the conformation of the ridge more compact and draws the loop containing Ala475 closer to hACE2. Figure 4 | Structure of SARS-CoV-2 chimeric RBD/human ACE2 complex. (a) Crystal structure of SARS-CoV-2 chimeric RBD/hACE2 complex. ACE2, RBD core, RBM, a retained side loop from SARS-CoV, and a zinc ion in ACE2 are presented in green, cyan, magenta, orange, and blue, respectively. (b) Conformation comparison between the ridges in SARS-CoV-2 RBM (purple) and SARS-CoV RBM (orange). (c) Comparison of conformations between the ridges from another view angle. In SARS-CoV RBM, a proline-proline-alanine motif is shown. In SARS-CoV-2 RBM, a newly formed hydrogen bond, Phe486, and some interactions between the ridge and the N-terminal helix of ACE2 are shown. Reproduced with permission from ref 19. Copyright 2020 Springer Nature. Download figure Download PowerPoint Another structural difference between the RBMs of SARS-CoV-2 and SARS-CoV occurs near the two virus-binding hotspots at the RBM/hACE2 interface.19 Two hotspots were identified on hACE2: hotspot-31, consisting of a salt bridge between Lys31 and Glu35, and hotspot-353, consisting of a bridge between Lys353 and ASP38.25 Upon virus binding, the hotspots are buried in hydrophobic environments, providing significant energy to fuel virus/receptor interaction. In the SARS-CoV/hACE2 complex, the structures of hotspot-31 and hotspot-353 are supported by Tyr442 and Thr487 on SARS-CoV, respectively (Figure 5a). In comparison, Leu442 and Asn501 from SARS-CoV-2 RBM, corresponding to Tyr442 and Thr487 from SARS-CoV RBM, respectively, provided less support for the hotspot structures. Consequently, hotspot-21 was rearranged, and Lys31 and Glu35 from hACE2 both formed hydrogen bonds with Gln493 from SARS-CoV-2 RBM. Hotspot-353 also takes a slightly different conformation, and Lys353 from hACE2 forms a hydrogen bond with the main chain of SARS-CoV-2 RBM while maintaining the salt bridge with Asp38 from hACE2 (Figure 5b). Both hotspots have adjusted to reduced support from nearby RBD residues, yet still, become well stabilized at the SARS-CoV-2/hACE2 interface. Figure 5 | (a) Structural analysis at the interface between SARS-CoV-2 RBM and human ACE2. (b) Structural analysis at the interface between SARS-CoV RBM and human ACE2. Reproduced with permission from ref 19. Copyright 2020 Springer Nature. Download figure Download PowerPoint Besides, based on sequence alignment of SARS-CoV-2 S with multiple related SARS-CoVs, Walls et al.20,26 revealed that SARS-CoV-2 S had a unique polybasic "Arginine-Arginine-Alanine-Arginine (RRAR)" furin protease cleavage site at the S1/S2 boundary (Figure 6). The furin cleavage site of SARS-CoV-2 was also confirmed by other research groups.27–30 In addition, it was reported that SARS-CoV-2 entry into host cells was dependent on the cleavage ability of cathepsins (e.g., cathepsin L and cathepsin B),31 which is involved with antigen processing during the viral infection, and transmembrane protease serine protease-2 (TMPRSS-2)32 that cleaves and activates the viral surface proteins. Therefore, the presence of the protease cleavage sites in S glycoprotein could facilitate the fusion between SARS-CoV-2 and host cells, and consequently, enhance viral entry in tissues. Based on numerous research studies, it can be deduced that the enhanced binding affinity to ACE2 and the presence of protease cleavage sites in S glycoprotein account for the rapid and virulent human-to-human transmission of SARS-CoV-2. Figure 6 | Sequence comparison of SARS-CoV-2 S glycoprotein with S glycoproteins from multiple SARS-CoVs and SARS-related CoVs (SARSr-CoVs) indicating that only SARS-CoV-2 spike contains a putative "RRAR" furin cleavage site at the S1/S2 boundary. Download figure Download PowerPoint Diagnosis of COVID-19 Compared with SARS-CoV, SARS-CoV-2 is characterized by more rapid spread and virulent human-to-human transmission. SARS-CoV-2 is airborne transmitted and causes infectious and deadly pneumonia, termed as COVID-19. Patients with COVID-19 usually have pneumonia-like symptoms, such as fever, dry cough, fatigue, sputum production, short breath, and sore throat. However, some patients with COVID-19 show mild or even no symptoms. These asymptomatic individuals are highly contagious. The World Health Organization (WHO) declared a COVID-19 as pandemic on March 11, 2020. As of July 29, 2020, the number of cases of COVID-19 infection had exceeded 16.5 million, with 655,112 deaths at an astonishing speed. To prevent the route of transmission and eliminate the source of infection, the best strategy for COVID-19 management is early detection, early isolation, and treatment of the infected. This leads us to a discussion of diagnostic methods and drug development. Computed tomography COVID-19 is an airborne respiratory disease, ultimately resulting in severe acute respiratory syndrome. Therefore, the clinical status of the lung of patients can provide important information for COVID-19 diagnosis. As a noninvasive imaging test based on the combination of X-ray, reconstruction mathematics, and computer technology, CT scan of the chest is expected to provide detailed pictures of lung abnormalities, particularly to a COVID-19 diagnosis.33 Up to now, numerous CT scans of COVID-19 patients have revealed the presence of ground-glass opacities (GGO), characterized by a peripheral and subpleural distribution, as the main CT feature of COVID-19 pneumonia (Figure 7).34–37 To explore the correlation between chest CT and reverse transcription-polymerase chain reaction (RT-PCR) testing in COVID-19, Ai et al.38 systematically analyzed their response to clinical manifestations in 1014 cases of suspected COVID-19. The results indicated that the favorable rates of RT-PCR assay and chest CT scans were 59% (601/1014) and 88% (888/1014), respectively. Given its sensitivity, a CT scan was adopted as a clinical criterion for the COVID-19 cases from Hubei Province of China in the revised fifth edition of the Guideline of Diagnosis and Treatment. Although the specificity of CT for COVID-19 is relatively low (∼25%) for overlapped imaging features with other viral pneumonia,38,39 chest CT scan undoubtedly plays a critical role in early identification, disease progression, and treatment monitoring of COVID-19 pneumonia, especially for the hospitals or communities lacking nucleic acid testing equipment and kits. Figure 7 | CT images from the survival group and mortality group. GGO with peripheral in and lung were in the CT images of a from the survival group with and GGO were observed in the CT images of a from the mortality group from ref Copyright 2020 The Download figure Download PowerPoint acid As the critical of SARS-CoV-2, RNA a significant molecular for COVID-19 diagnosis. in nucleic acid sequencing the and of viral RNA in January 2020, the genome sequences of SARS-CoV-2 were identified from infected Sequence information was in with the of on January 2020. The diagnostic were provided for SARS-CoV-2 in the sequence information and diagnostic was critical for the spread of SARS-CoV-2. 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