| Coronavirus Disease 2019(COVID-19) is an acute respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Since the outbreak of COVID-19 in2019,it has caused at least 760 million infections and more than 6.8 million deaths worldwide.The spike(S)protein of SARS-CoV-2 is a key protein that determines the pathogenicity and transmissibility of the virus.The extracellular domain of the S protein contains the “RRAR” motif,which can be recognized and cleaved into the S1 and S2 subunits by a calcium-dependent proprotein invertase called furin protease.The deletion or mutation of the furin cleavage site of the S protein will significantly inhibit the virulence and infectivity of SARS-CoV-2,indicating that furin is very important for the infection of SARS-CoV-2and has the potential to be used as a target for antiviral drugs.The regulation mechanism of furin’s enzymatic activity under physiological or viral infection conditions is still unclear.Therefore,exploring the host factors related to the regulation of furin activity and clarifying the mechanism of regulating furin enzyme activity will provide a theoretical basis for the development of furin-related antiviral drugs.This study found that the soluble N-ethylmaleimide-sensitive factor attachment protein alpha(α-SNAP),which is mainly involved in vesicle transport mechanisms,interacts with the SARS-CoV-2 S protein through mass spectrometry analysis.When verifying the interaction between α-SNAP and SARSCoV-2 S protein,we found that the cleavage of S protein was significantly inhibited when α-SNAP was co-expressed with SARS-CoV-2 S,and the effect of inhibiting the cleavage of S protein was gradually enhanced with the increasing expression of α-SNAP,indicating that α-SNAP could inhibit the cleavage of SARS-CoV-2 S;Co-expressed of NSF(N-ethylmaleimide-sensitive fusion factor),another partner molecule involved in the formation of 20 S complex,with SARS-CoV-2 S revealed that the cleavage of S was not affected.This result suggest that α-SNAP inhibits the cleavage of SARS-CoV-2 S independent of the function of the 20 S complex;A series of α-SNAP truncated mutants were co-expressed with SARSCoV-2 S,and found that the 27/28 and 231-250 amino acids are the key regions for inhibiting the cleavage of S protein by α-SNAP;Further study found that α-SNAP also inhibit cleavage of the furin-dependent Middle east respiratory syndrome coronavirus,Zaire ebola virus and Marburg virus glycoproteins,but not furin-independent SARS-CoV S proteins,it is suggested that α-SNAP inhibition of SARS-CoV-2 S cleavage may be related to furin.In order to elucidate mechanism of α-SNAP inhibition of SARS-CoV-2 S cleavage,coimmunoprecipitation and Pull-down experiments were performed,and the results showed that α-SNAP directly interacted with furin,and the 27/28 and 231-250 amino acids of α-SNAP and the P domain of furin are the key regions for their interaction.Furin enzyme activity assay showed that overexpression ofα-SNAP could significantly inhibit furin enzyme activity in cells and cell supernatant,reducing α-SNAP expression significantly promoted furin enzyme activity in cells and cell supernatant,and 27/28 and 231-250 amino acids were the key regions that overexpression of α-SNAP inhibited furin enzyme activity;The experimental results of viral infection showed that SARS-CoV-2 infection could promote the expression of α-SNAP,and overexpression of α-SNAP significantly inhibited the internalization of SARSCoV-2,and inhibiting the viral infection;Interferon stimulation experiments showed that type I or III interferon significantly promoted the expression of α-SNAP,but type II interferon did not promote the expression of α-SNAP,indicating that α-SNAP was an interferon-stimulating gene(ISG).In summary,this study revealed the new function of α-SNAP,and found that α-SNAP is a novel ISG whose expression is regulated by type I and type III interferons.The up-regulated expression of α-SNAP can inhibit the enzyme activity of furin through direct interaction with furin,and inhibit the cleavage and maturation of various viral membrane proteins such as SARS-CoV-2 S,thus affecting the invasion of viruses.This study provides a new target for the prevention and treatment of furin-dependent viral diseases such as SARS-CoV-2 and the development of furin inhibitors. |
