Study On The Regulation And Mechanism Of Intracellular Trafficking Of SARS-CoV-2 S Protei

BACKGROUND:The Coronavirus Disease(COVID-19)caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is one of the most serious diseases in human history.The development of drugs targeting key viral proteins in the life cycle of the novel coronavirus is a research focus for combating COVID-19.The novel coronavirus has undergone several evolutions and global pandemic,and these mutant strains have brought significant challenges to the fight against COVID-19.The mainstream view is that the evolution of the novel coronavirus is driven by mutations in its key structural proteins,particularly the Spike Protein(S).As one of the core drug targets for combating COVID-19,the S protein is the basis for neutralizing antibodies and vaccines,especially mRNA vaccines,which have been the most successful antiviral strategies in clinical practice.Neutralizing antibodies need to recognize the S protein exposed on the surface of infected host cells and clear the infected host cells through antibody effector functions.Therefore,the cell surface exposure of the S protein is necessary for neutralizing antibodies to fully exert their antiviral function.At the same time,the mRNA vaccine based on the S protein as an antigen also needs to expose the S protein on the surface of antigen-presenting cells to more effectively activate B cells,initiate the adaptive immune process,and ultimately produce long-term memory B cells that can secrete neutralizing antibodies.The COPⅠ-mediated sorting of S depends on the KxHxx motif in the C-terminus of the S protein,which is recognized by α-and P’-COP.Due to the weak COPⅠ vesicle transport sorting mechanism of S,the vast majority of S protein is localized in early secretory organelles,and only a small proportion is leaked and released to the cell surface.Based on the background above,this article designs a COPⅠ sorting inhibitor that can block the sorting of S protein by COPⅠ,promote more secretion of S protein to the cell surface,and enhance the function of neutralizing antibodies and vaccines.This study is also the first attempt to design drugs targeting COPⅠ substrate sorting.This is achieved on the basis of systematic elucidation of the structural biology and biochemical characterization of S protein being sorted by COPⅠ.OBJECTIVE:Regulate the intracellular transport of the SARS-CoV-2 S protein,explore the dynamic molecular processes of intracellular retention and transport,provide potential drugs for antiviral therapy,and offer new avenues for studying the evolutionary mutations of the S protein.METHODS:In order to design inhibitors that block the COPⅠ sorting of S and study the potential function of S protein mutations,a series of research methods were used in this paper including:(1)Biochemical characterization of the interaction between S and COPⅠ,such as affinity assays and mutation analysis.(2)Structural biology methods were used to systematically analyze the crystal structures of the complexes formed by multiple COPⅠ sorting signals binding to COPⅠ,as well as the crystal structure of COP Ⅱ sorting signal binding to COP Ⅱsubunits,within the human coronavirus family.(3)Design and activity testing of peptide-based COPⅠ substrate sorting inhibitors.By combining the structural features of COPⅠ substrate recognition,a high-affinity chimeric peptide TAT-WDM was designed to inhibit COPⅠ substrate recognition.This peptide has the ability to penetrate cell membranes.Its activity was confirmed,which can promote the presentation of S protein on the host cell surface,and its complex crystal structure with COPⅠ was also analyzed.(4)Using cellular and molecular biology methods to investigate the effect of the chimeric peptide TAT-WDM on promoting the exposure of S protein on the cell membrane surface.A stable cell line expressing S protein was constructed,and methods such as immunofluorescence,flow cytometry,cell surface protein biotinylation,Western blot,and cell fusion were used to determine the regulatory effect of TAT-WDM on intracellular S protein and to detect the cytotoxicity of TAT-WDM on cells.(5)Using the peptide TAT-WDM as a probe,the region responsible for intracellular retention of S protein was identified through immunofluorescence,and the proteins involved in the intracellular localization of the S protein in the new COVID-19 variants were determined through co-immunoprecipitation(Co-IP).RESULT:(1)Multiple coronaviruses’ C-terminal sequences were compared,and peptides containing the aforementioned sequence were synthesized.The dissociation constant(KD)between the new coronavirus S protein and COPⅠ was measured by using isothermal titration calorimetry(ITC)and fluorescence polarization(FP).The value is about ImM.The dissociation constants between the S proteins of other coronaviruses and COPⅠ were mostly in the range of 100-500μM.(2)Using structural biology methods,the complex crystal structures of several COPⅠ sorting signals binding to COPⅠ in the human coronavirus family were systematically analyzed.It was found that the S protein binds to the β’-COP subunit of COPⅠ through different sorting signals(KxHxx,KxxHxx,and Hxx motifs)with similar modes of action.The binding site 1 onβ’-COP consists of Arg15,Arg272,and Lys17,while the binding site 2 is composed of Asp 117,D98,and D206.The K residue of the dilysine motif is oriented towards these two COPⅠ binding sites,causing KKxx to bind to the β-propeller domain of β’-COP in a clockwise direction and KxKxx to bind in a counterclockwise direction.The interaction mode between the KxHxx motif of the S protein of the novel coronavirus and the β-propeller domain is very similar to that of KxKxx binding to the β-propeller domain.The S protein of SARS-CoV-2 contains a sorting signal,xD,that binds to the Sec24A subunit of COPⅡ in a manner similar to the classic DxE motif.The two C-terminal aspartic acid residues of the xD motif participate in the interaction with key amino acids R430,Y437,R750,and R752 of the COPⅡ binding site.(3)Based on the structure and affinity characteristics of S protein binding to COPⅠ,a chimeric peptide TAT-WDM with strong COPⅠ binding and significant cellular uptake ability was designed and optimized.The dissociation constant between the peptide and COPⅠ was 1.8 μM.Competition binding experiments showed that TAT-WDM can significantly inhibit the binding of S protein to COPⅠ.At the same time,it was observed that TAT-WDM can significantly penetrate the cell membrane.Subcellular localization experiments showed that it is consistent with the location of COPⅠ vesicle budding,indicating that TAT-WDM has the basic conditions to become a COPⅠ inhibitor:it can enter the cell while inhibiting the binding of S to COPⅠ.The crystal structure of the TAT-WDM complex binding to COPⅠ was analyzed,and it was found that its interaction mode was similar to that of the WDM-COPⅠ complex,proving that the fusion of TAT did not change the binding characteristics of WDM.(4)Stable cell lines expressing the S protein were constructed,and experiments at the cellular level showed that the use of TAT-WDM significantly promotes the secretion of the S protein on the cell membrane surface.The S protein on the cell surface can fuse with cells expressing the ACE2 receptor to form syncytia.In the cell fusion assay,the use of TAT-WDM promoted the formation of more syncytia,indicating that more S protein reached the cell surface.The same result was observed in the presence of S,M,and E proteins.In the antibody-dependent cell-mediated cytotoxicity(ADCC)assay,S protein on the cell surface is recognized by neutralizing antibodies and targeted for killing by NK cells.The use of TAT-WDM significantly enhances this killing effect.Meanwhile,in cell toxicity assays,TAT-WDM is found to have extremely low cytotoxicity as a novel COPⅠ sorting inhibitor,demonstrating its potential as a drug with high safety.(5)TAT-WDM promotes intracellular trafficking of S protein of novel coronavirus variants.The cellular retention ability of the S protein varies among different variants of the novel coronavirus,with the Omicron BA.1 variant showing a significantly stronger cellular retention ability than previous variants.Using Alpha variant B.1.1.7 S protein as control,chimeric S protein were constructed by introducing the mutations on the Omicron BA.1 variant S protein into the S protein of the Alpha variant B.1.1.7.We then used immunofluorescence to measure the fluorescence intensity of the chimeric S protein on the cell surface,in order to identify which mutation sites can affect the exposure of S protein on the cell surface and to explore the intracellular retention mechanism of Omicron variant.The results showed that two mutation sites in the Nterminal domain and one in the receptor-binding domain caused stronger ER retention of the S protein in the BA.1 strain.The mutation of the S protein results in its misfolding,thus preventing it from being transported out of the endoplasmic reticulum.The results of immunoprecipitation also reflected the interaction between Bip(an ER stress marker)and calnexin with the misfolding S protein,which caused the ER retention of the S protein and led to stronger ER stress.Comparing with the sequences of Omicron sub-lineages,it was found that they all contain the mutation regions identified in this study.Such results help to explain the pathogenic changes caused by S protein mutations in the Omicron strain.CONCLUSION:In this paper,the crystal structure of multiple COPⅠ sorting signals of the human coronavirus family bound to COPⅠ were first analyzed and COPⅠ substrate sorting inhibitor TAT-WDM bound to COPⅠ as well.The measurement of the affinity between the Cterminal sorting signal of the S protein and COPⅠ revealed that the affinity between the S protein and COPⅠ is relatively weak,which means that the S protein is partially transported to the cell membrane surface.Based on the inspiration that S protein can be exposed on the cell surface,a S protein COPⅠ sorting inhibitor was developed.The inhibitor can compete with COPⅠ for binding to S protein,regulate the intracellular transport of S protein,and promote more S protein secretion to the cell surface.This can then enhance the clearance of infected cells through the ADCC effect mediated by S protein neutralizing antibodies.In addition,it has also been found that the S protein of the Omicron BA.1 variant of the coronavirus is more tightly retained in the endoplasmic reticulum than the S protein of the original strain,which is caused by the interaction between the mutated S protein and endoplasmic reticulum molecular chaperones.At the same time,the mutation sites that may cause intracellular retention of the S protein have been identified.The findings of this study not only demonstrate that COPⅠ is a potential drug target against COVID-19,providing potential drugs for antiviral therapy,but also provide some insights into understanding the evolution of SARS-CoV-2 driven by S protein mutations.Understanding the signal that affects the intracellular retention of S protein in the Omicron variant can provide insights for the design of mRNA vaccines against COVID-19.In the future,small molecules that inhibit COPⅠ transport can be screened from traditional Chinese medicine,thereby expanding the application of TCM in combating the virus and promoting the development of TCM.