SARS-CoV Nonstructural X2 Protein Localizes In Nucleolus And Induces Cell Cycle Arrest And Apoptosis In Transfected Cells

SARS is a system disease that injures many organs, such as lung, liver, kidney, and immune organe, and the respiratory distress and decreased immune function are proposed to be the main causes of death. SARS-CoV, cause of the life-threatening atypical pneumonia, is not closely related to any of the previously characterized coronavirus. The genome of SARS-CoV codes replicase and four major structural proteins, which are common to all known coronavirus, and a number of uncharacter proteins. Published studies suggest that some uncharacter proteins may have important roles in the replication, virulence and pathogenesis of viruses. Among the potential SARS-CoV uncharacter proteins, X2 protein (ORF4) is composed of 154 amino acids, lacking significant similarities to any known proteins. Till now, there is no reported study about the function of X2 protein. In this study, X2 cDNA was linked with the EGFP and myc tags at the N and C terminus, respectively, and transfected into COS-7 cells. Immunofluorescence staining of the expressed tagged X2 protein showed that X2 protein was mainly localizated in the nuclear. Further co-localization with C23/nucleolin antibody demonstrated that X2 protein localized in the nucleolus of transfected cells in the absence of any other viral proteins and displayed dotted nucleolus signal pattern. The localization pattern of myc-X2 and EGFP-X2 fusion proteins were similar to that in transfected Vero, 293 and COS-7 cells of X2-EGFP, and X2 protein was mobile freely to the nucleolus from 12h after transfection. By using a series of X2-truncated mutants fused with EGFP, a nucleolus localization signal from amino acid 134 to 154 was identified, which was consistent with that of bioinformatic analysis.Over-expression of X2-EGFP protein could block cell cycle progression at G₀/G₁ phase in Vero, 293 and COS-7 cells, and especially induce apoptosis in COS-7 cells. These results is provides a new insight for further study of X2 protein on the pathogeny ofSARS-CoV infection.