Basis Data Jurnal

[Sherry Galeazzi]

Penelusuranliteratur idealnya dilakukan pada basis data (database) kedokteran elektronik, bukan pada satu layanan jurnal tertentu, dengan menggunakan mesin pencari elekronik (search engine).Berikut merupakan daftar database jurnal kesehatan / kedokteran beserta lembaga pengembangnya.Read less

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Receptor recognition by SARS-CoV has been extensively studied. A virus-surface spike protein mediates the entry of coronavirus into host cells. The spike protein of SARS-CoV contains a RBD that specifically recognizes ACE2 as its receptor3,4. A series of crystal structures of the SARS-CoV RBD from different strains in complex with ACE2 from different hosts has previously been determined3,11,12. These structures showed that SARS-CoV RBD contains a core and a receptor-binding motif (RBM); the RBM mediates contacts with ACE2. The surface of ACE2 contains two virus-binding hotspots that are essential for SARS-CoV binding. Several naturally selected mutations in the SARS-CoV RBM surround these hotspots and regulate the infectivity, pathogenesis, and cross-species and human-to-human transmissions of SARS-CoV3,11,12.

a, Crystal structure of the SARS-CoV-2 chimeric RBD complexed with ACE2. ACE2 is shown in green. The RBD core is shown in cyan. The RBM is shown in magenta. A side loop in RBM is shown in orange. A zinc ion in ACE2 is shown in blue. b, Comparison of the conformations of the ridge in SARS-CoV-2 RBM (magenta) and SARS-CoV RBM (orange). c, Comparison of the conformations of the ridge from another viewing angle. In the SARS-CoV RBM, a proline-proline-alanine motif is shown. In the SARS-CoV-2 RBM, a newly formed hydrogen bond, Phe486, and some of the interactions of the ridge with the N-terminal helix of ACE2 are shown.

A marked structural difference between the RBMs of SARS-CoV-2 and SARS-CoV is the conformation of the loops in the ACE2-binding ridge (Fig. 1b, c). In both RBMs, one of the ridge loops contains an essential disulfide bond and the region between the disulfide-bond-forming cysteines is variable (Fig. 1c and Extended Data Fig. 1). Specifically, human and civet SARS-CoV strains and bat coronavirus Rs3367 all contain a three-residue motif proline-proline-alanine in this loop; the tandem prolines allow the loop to take a sharp turn. By contrast, SARS-CoV-2 and bat coronavirus RaTG13 both contain a four-residue motif glycine-valine/glutamine-glutamate/threonine-glycine; the two relatively bulky residues and two flexible glycines enable the loop to take a different conformation (Fig. 1c and Extended Data Fig. 1). Because of these structural differences, an additional main-chain hydrogen bond forms between Asn487 and Ala475 in the SARS-CoV-2 RBM, causing the ridge to take a more compact conformation and the loop containing Ala475 to move closer to ACE2 (Fig. 1c). As a consequence, the ridge in the SARS-CoV-2 RBM forms more contacts with the N-terminal helix of ACE2 (Extended Data Fig. 4b). For example, the N-terminal residue Ser19 of ACE2 forms a new hydrogen bond with the main chain of Ala475 of the SARS-CoV-2 RBM, and Gln24 in the N-terminal helix of ACE2 also forms a new contact with the SARS-CoV-2 RBM (Fig. 1c and Extended Data Fig. 4b). Moreover, compared with the corresponding Leu472 of the SARS-CoV RBM, Phe486 of the SARS-CoV-2 RBM points in a different direction and inserts into a hydrophobic pocket involving Met82, Leu79 and Tyr83 of ACE2 (Figs. 1c, 2a, b). In comparison to the SARS-CoV RBM, these structural changes in the SARS-CoV-2 RBM are more favourable for ACE2 binding.

Having compared ACE2 recognition by SARS-CoV-2 and SARS-CoV, we further investigated human ACE2 binding by bat RaTG13. To this end, we performed a pseudovirus entry assay in which retroviruses pseudotyped with RaTG13 spike (that is, RaTG13 pseudoviruses) were used to enter ACE2-expressing human cells (Fig. 3b). The results showed that RaTG13 pseudovirus entry into the cells depends on ACE2. Additionally, RaTG13 spike was not cleaved on the pseudovirus surface. SARS-CoV-2 pseudovirus entry also depends on ACE2, but its spike was cleaved to S2 on the pseudovirus surface (probably because of a furin site insertion16) (Fig. 3b). Moreover, we performed a protein pull-down assay using ACE2 as the bait and cell-associated RaTG13 spike as the target (Fig. 3c). We found that the RaTG13 spike was pulled down by ACE2. Therefore, similar to SARS-CoV-2, bat RaTG13 binds to human ACE2 and can use human ACE2 as its entry receptor.

The current SARS-CoV-2 outbreak has become a global pandemic. Previous structural studies on SARS-CoV have established receptor recognition as an important determinant of SARS-CoV infectivity, pathogenesis and host range9. On the basis of the structural information presented here, along with biochemical data, we discuss the receptor recognition and evolution of SARS-CoV-2.

Next, we investigated how SARS-CoV-2 may have been transmitted from bats to humans. First, we found that bat RaTG13 uses human ACE2 as its receptor (Fig. 3b, c), suggesting that RaTG13 may infect humans. Second, as with SARS-CoV-2, bat RaTG13 RBM contains a similar four-residue motif in the ACE2-binding ridge, supporting the notion that SARS-CoV-2 may have evolved from RaTG13 or a RaTG13-related bat coronavirus (Extended Data Table 3 and Extended Data Fig. 7). Third, the L486F, Y493Q and D501N residue changes from RaTG13 to SARS-CoV-2 enhance ACE2 recognition and may have facilitated the bat-to-human transmission of SARS-CoV-2 (Extended Data Table 3 and Extended Data Fig. 7). A lysine-to-asparagine mutation at the 479 position in the SARS-CoV RBD (corresponding to the 493 position in the SARS-CoV-2 RBD) enabled SARS-CoV to infect humans3. Fourth, Leu455 contributes favourably to ACE2 recognition, and it is conserved between RaTG13 and SARS-CoV-2; its presence in the SARS-CoV-2 RBM may be important for the bat-to-human transmission of SARS-CoV-2 (Extended Data Table 3 and Extended Data Fig. 7). Host and viral factors other than receptor recognition also have important roles in the cross-species transmission of coronaviruses20,21. Nevertheless, the identified receptor-binding features of the SARS-CoV-2 RBM may have facilitated SARS-CoV-2 to transmit from bats to humans (Extended Data Fig. 7).

Finally, this study helps to inform intervention strategies. First, neutralizing monoclonal antibodies that target the SARS-CoV-2 RBM can prevent the virus from binding to ACE2, and are therefore promising antiviral drugs. Our structure has laid out all of the functionally important epitopes in the SARS-CoV-2 RBM that can potentially be targeted by neutralizing antibody drugs. Thus, this study can help to guide the development and optimization of these antibody drugs. Second, the RBD itself can function as a subunit vaccine10,23. The functionally important epitopes in the SARS-CoV-2 RBM that were identified in this study can guide structure-based design of highly efficacious RBD vaccines. Such a structure-based strategy for subunit vaccine design has previously been developed24. This strategy may be helpful in designing SARS-CoV-2 RBD vaccines. Overall, this study can help to inform structure-based intervention strategies that target receptor recognition by SARS-CoV-2.

All of the proteins were prepared from Sf9 insect cells using the Bac-to-Bac system (Life Technologies) as previously described3. In brief, the His6-tagged proteins were collected from cell culture medium, purified using a Ni-NTA column, purified further using a Superdex200 gel filtration column (GE Healthcare) and stored in a buffer containing 20 mM Tris pH 7.2 and 200 mM NaCl. The Fc-tagged protein was purified in the same way as the His6-tagged proteins, except that the protein A column replaced the Ni-NTA column in the procedure.

The SPR assays using a Biacore 2000 system (GE Healthcare) were carried out as described previously12. In brief, different RBDs were covalently immobilized to a CM5 sensor chip through their amine groups (GE Healthcare). The running buffer contained 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% Tween-20. Serial dilutions of purified recombinant ACE2 were injected ranging in concentration from 5 to 80 nM for the SARS-CoV-2 RBD and chimeric RBD, and from 20 to 320 nM for the SARS-CoV RBD. The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software (GE Healthcare).

J.S. conceptualized the project, expressed and purified proteins, performed crystallization, carried out protein pull-down experiments and the pseudovirus entry assay, and reviewed the manuscript. G.Y. performed crystallization, determined and refined the structure, analysed the structure, performed the SPR experiment, and reviewed the manuscript. K.S. collected X-ray diffraction data, determined and refined the structure, analysed the structure, and reviewed the manuscript. Y.W. conceptualized the project, expressed and purified proteins, performed protein pull-down experiments and the pseudovirus entry assay, and reviewed the manuscript. C.L. performed protein pull-down experiments and the pseudovirus entry assay, and reviewed the manuscript. H.A. provided resources, analysed the structure, and reviewed the manuscript. Q.G. performed protein pull-down experiments and the pseudovirus entry assay, and reviewed the manuscript. A.A. expressed and purified proteins, and reviewed the manuscript. F.L. conceptualized and supervised the project, provided resources, guided the experiments and data analysis, and wrote the manuscript.

RBM is shown in magenta. Previously identified critical ACE2-binding residues are shown in blue. The seven RBM residues that differ between the SARS-CoV-2 wild-type RBD and SARS-CoV-2 chimeric RBD are shaded. A critical arginine on the side loop of the SARS-CoV RBM that forms a strong salt bridge with ACE2 is shown in green. Another arginine in the core structure that interacts with glycan is shown in cyan. The residues on the variable loop between two disulfide-bond-forming cysteines in the ACE2-binding ridge are shown in red. The important motif changes in the ACE2-binding ridge are underlined. GenBank accession numbers are: QHD43416.1 for SARS-CoV-2 spike; AFR58742 for human SARS-CoV spike; AY304486.1 for civet SARS-CoV spike; MG916901.1 for bat Rs3367 spike; QHR63300.2 for bat RaTG13 spike. Two coronaviruses, CoV-pangolin/GD and CoV-pangolin/GX, were isolated from pangolins at two different locations in China, Guangdong and Guangxi; their RBD sequences were from a previous study22.

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