| Research BackgroundSevere acute respiratory syndrome coronavirus 2(SARS-Co V-2),has been an epidemic for nearly three years,posing a major threat to human lives,property,and health,and causing huge losses to the economy around the world.As of March 7,2023,coronavirus disease 2019(COVID-19),caused by SARS-Co V-2,had resulted in more than 750 million of patients,leading to about 6.86 million deaths around the world.Among the species that could be infected with SARS-Co V-2,mink,hamster,and whitetailed deer could be transmitted not only within the species but also back to humans.Therefore,more and more attention had been paid to the infectivity of SARS-Co V-2 in animals at home and abroad.Mice were known hosts of a number of infectious diseases,such as pestis,epidemic hemorrhagic fever.Considering that mice were widely existing and hard to be controlled,particularly in communities of poor sanitation,and may cause catastrophic impacts if being a SARS-Co V-2 transmitter.Studies had shown that the N501 Y mutation in the spike protein of SARS-Co V-2 could break the cross-species transmission barrier,allowing SARS-Co V-2 to establish effective infection in mouse.In this situation,it was essential to investigate which mutation sites in natural mutations could cause cross-species transmission of SARS-Co V-2 to mice and the possible mechanisms.In addition,the relationship between the parsed mutant loci and the infectivity of SARS-Co V-2 endemic strains to mice,such as Omicron strains,was verified,and the early warning of the risk of cross-species transmission of SARS-Co V-2endemic strains were provided either.Research ObjectivesSARS-Co V-2 genomic RNA had undergone frequent mutations as the COVID-19 pandemic drags on,leading to various variants.Some of variants enhanced the ability to spread across species,making the intermediate host become a new source of transmission and leading to the risk of another outbreak.It had been shown that N501 Y mutations could mediate SARS-Co V-2 infection in mice and that mutations within the receptor binding domain(RBD)had an enhanced effect on the ability of N501 Y to mediate the cross-species transmission of SARS-Co V-2 to mice,but it was not clear whether mutations outside the RBD also have this function,so this study was intended to explore the effect of non-RBD mutations on the ability of N501 Y to mediate the cross-species transmission of SARS-Co V-2 to mice and the related mechanisms.Research Methods1.Screening of mutant loci for cross-species transmission of SARS-Co V-2 to mice:Currently,there were mouse-adapted strains obtained by artificially domesticating SARS-Co V-2 wild strains by successive generations in mice,by reverse genetics techniques and by identifying nature SARS-Co V-2 variants.The mutations on the mouse-adapted strains of SARS-Co V-2 were compared and analyzed through literature research to screen for potential cross-species transmission loci.2.SARS-Co V-2 infectivity analysis:(1)Construction of S protein expression vectors: S protein expression vectors for SARS-Co V-2 mouse-adapted strain and its related mutants were constructed by restriction endonuclease digestion,point mutation,homologous recombination,and other molecular biology methods,and the correct target plasmids were selected through sequencing.(2)Infectivity analysis: S protein expression vectors of SARS-Co V-2 mouse-adapted strain and its related mutants were transfected into 293 T cells stably expressing human angiotensin-converting enzyme 2(h ACE2)and 293 T cells stably expressing mouse angiotensin converting enzyme2(m ACE2)by transfection reagents,and wild-type SARS-Co V-2 S protein was used as a control.The membrane fusion ability of the mouse-adapted strain and its related mutants spike were examined by employing the syncytium formation assay.The production of S2 subunit was detected using western bolt(WB).The binding affinity of S protein with the receptor was detected using immunoprecipitation(IP).Pseudoviruses of SARS-Co V-2 mouse-adapted strain and related mutants were packaged using retroviral three-plasmid packaging system and the infection level was assessed by firefly luciferase reporter gene assay after infection with cells.3.Mechanism analysis of SARS-Co V-2 cross-species transmission: S protein was the key protein for SARS-Co V-2 to invade host cells,and its interaction with receptor ACE2 would be changed after mutation of S protein.The revealed SARS-Co V-2 S protein structure was used as a template for 3D structure modeling using the 3D algorithm of SWISS-MODEL,and the effect of viral mutation on viral transmission ability was analyzed in the 3D structure.4.Validation of SARS-Co V-2 endemic strains: SARS-Co V-2 endemic strains associated with the parsed mutant loci were selected for validation.The expression vector of S protein of SARS-Co V-2 endemic strain were obtained by restriction endonuclease digestion,point mutation,homologous recombination,and other molecular biological means.The correct target plasmids were selected by sequencing.The mutation sites were subjected to revertant mutagenesis,changes in membrane fusion ability were detected by syncytium formation assay,and changes in infectivity were assessed by pseudovirus infection assay.Therefore,the study could provide theoretical support and early warning on whether the SARS-Co V-2 endemic strain has the risk of cross-species transmission to mice.Research Results1.The mutation loci of the SARS-Co V-2 mouse-adapted strain were analyzed comparatively by literature research,one mouse-adapted strain MA-SARS2 and two non-RBD mutation loci: Ins215 KLRS and H655 Y were obtained by screening and analysis.2.Regarding the results of SARS-Co V-2 infectivity analysis:(1)SARS-Co V-2mouse-adapted strain MA-SARS2 possessed infectivity for 293T-m ACE2 cells,while there was no decrease in infectivity for 293T-h ACE2 cells.(2)Syncytium formation assay and pseudovirus assay results demonstrated that Ins215 KLRS and H655 Y could assist N501 Y to improve the infectivity of the virus to m ACE2-expressing cells.(3)By WB,Ins215 KLRS and H655 Y were found to increase the production of S2 subunit in293T-m ACE2 cells,and IP experiments demonstrated that Ins215 KLRS and H655 Y could assist N501 Y to enhance receptor binding.3.The results of studies on the resolution of the cross-species transmission mechanism of SARS-Co V-2(1)H655Y mutation disrupted the interaction between H655 and N657,leading to a looser loop wrapping the furin cleavage finger that protruding through the loop,which conceivably facilitates the efficiency of furin protease recognition and cleavage to promote host entry.(2)Ins215KLRS enhanced the interactions inner S protein monomer and weakened the monomer-monomer interactions,resulting in a more open S protein.It was expected to promote receptorbinding-induced shielding of S1,thus unsheathing the fusogenic S2 to mediate host entry.4.Research results on the validation of the SARS-Co V-2 endemic strain:(1)The H655 Y mutation was found to be the key mutation for the high infectivity of the Gamma variant in 293T-m ACE2 cells by pseudovirus assay.(2)Ins214EPE and H655 Y were found to be key mutations for the high infectivity of the Omicron variant in 293 Tm ACE2 cells by syncytium formation assay and pseudovirus assay.Research ConclusionsIn this study,Results showed that SARS-Co V-2 S protein non-RBD mutations Ins215 KLRS and H655 Y could assist N501 Y to enhance mouse infectivity by infectivity analysis.Meanwhile,Ins215 KLRS and H655 Y mutations increased the production of S2 subunit and enhanced receptor binding.Structural analysis revealed that mutation of the H655 site to Y655 disrupted the interaction with N657,leading to a looser loop wrapping the furin cleavage finger that protruded through the loop.Ins215 KLRS enhanced the interaction inner S protein monomer and weakened the monomer-monomer interaction,resulting in a more open S protein.Further validation revealed that H655 Y was critical for mouse infection of the Gamma variant,and Ins214 EPE and H655 Y were critical for mouse infection of the Omicron variant.The above results provided a proof-of-concept example for long-range regulation of RBDmediated infectivity by non-RBD mutations,which is an important guide to explain the high infectivity of Omicron and other SARS-Co V-2 variants in mice,and this should be taken into account during the prevention of emerging SARS-Co V-2 variants such as Omicron. |
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