Studies On The Structure And Function Of SARS-CoV-2 Open Reading Frame-7a

At the end of 2019,a health emergency event that was induced by severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)broke out in Wuhan,China.The World Health Organization named the infection event a coronavirus disease-2019(COVID-19).The patients infected with SARS-CoV-2 usually have been treated by lopinavir,remdesivir and hydroxychloroquine in clinical treatment.These anti-viral drugs could inhibit the replication of viruses in host cells through focused on mitochondria-centered cellular innate immunity,and cleared invasive virus rely on the up-regulation of interferon(IFN).However,the clinical treatment effect is very limited according to the research dates that from experimental group and the control group.Therefore,while improving herd immunity in healthy residents by strengthening development of vaccine research,effective antiviral drugs are also needed to participate in the treatment of clinical infected patients.Non-structure proteins(NSPs)have assisted with the replication of coronavirus rely on regulating the innate immune of host cells according to previous research.Nevertheless,recent studies showed that the viral open reading frames(ORFs)were abundantly expressed during virus infection.Among the ORFs family proteins,ORF7 a has been reported that being involved in replication and budding of SARS-CoV-2,but the special mechanism is unclear.Hence,revealing the structural features and biological functions of ORF7 a,which new ideas for the development of therapeutic drugs and clinical treatment strategies for SARS-CoV-2.First of all,the protein polarity analysis was used in this study to analyze that ORF7 a belongs to an unstable hydrophobin according to the amino acid sequence of ORF7 a.And the transmembrane region in its amino acid sequence was predicted.In addition,the ORF7 a transmembrane structure model was successfully constructed and evaluated.Subsequently,we predicted that ORF7 a localizes in the endoplasmic reticulum and interacts with sequestosome-1(SQSTM1,p62)and calnexin(CANX)through the subcellular localization and interaction network.Therefore,we speculate that ORF7 a was located in the endoplasmic reticulum and involved in modification of host cellular proteins,Ubiquitination-dependent degradation and autophagy.Next,in order to clarify the target of ORF7 a in the autophagy signaling pathway,the autophagic inhibitors were used to prevent the fusion between autophagosomes with lysosomes and conversion of LC3-Ⅰ to LC3-Ⅱ,including chloroquine(CQ),3-methyladenine(3-MA).And then,our results found that the plasmid of ORF7 a would aggravate the accumulation of p62 after the autophagy-lysosome pathway was blocked by CQ in the autophagy-deficient cell model.This study demonstrated that ORF7 a played the positively synergistic with CQ in physiological function and participated in the inhibition of autophagy-lysosome pathway.Moreover,we proved that ORF7 a could antagonize the up-regulation of autophagy that induced by rapamycin(RAPA).Our evidence revealed that ORF7 a was involved in inhibiting the autophagy-lysosomal pathway in host cells as an autophagy inhibitor.Finally,in order to further investigate the target of ORF7 a,we verified that ORF7 a could induce the up-regulation of p62 and the aggregation of fluorescent spots when the autophagy signaling pathway is blocked.According to the results of IF,we revealed the up-regulation and recruitment function of ORF7 a to p62.In addition,the results of the double fluorescence staining method showed that ORF7 a co-localized with p62 in the cytoplasm after transfection and expression in Hela cells.This evidence revealed that p62 was the target of ORF7 a on autophagy-lysosome pathway.This observation,in combination with a range of results of this study,we revealed that ORF7 a prevented the fusion of autophagosome with lysosome by binding and inhibiting the activity of p62.This mechanism could assist SARS-CoV-2 escape the digestive pathway that mediated by the autophagy-lysosome of host cells.Therefore,our results showed that ORF7 a may become a new drug target for the treatment of patients with SARS-CoV-2infection.