The subcellular location of transmissible gastroenteritis virus protein ORF7 and its effect on viral replication

ABSTRACT

The objective of the present study was to explore the subcellular localization of TGEV ORF7 and its effect on the viral replication. In this study, the TGEV ORF7 gene was amplified by RT-PCR. The eukaryotic expression vectors pGFP-ORF7 (GFP fused with the C-terminal of TGEV ORF7) and pORF7-GFP (GFP fused with the N-terminal of ORF7) were constructed by inserting the TGEV ORF7 gene into the pEGFP-C1 and pEGFP-N1 vectors. Then the two obtaining recombinant plasmids were transfected into the ST cells and the expression of the TGEV ORF7 protein was verified by western blot. And the subcellular localization of the ORF7 protein was confirmed by combining the bioinformatics and the laser confocal scanning microscopy methods together. Furthermore, the effect of ORF7 protein on TGEV replication was assessed by observation of cytopathic effect (CPE) and quantitative analysis of real-time PCR. The results showed that the recombinant plasmids pGFP-ORF7 and pORF7-GFP were constructed successfully. Western blot analysis showed that the GFP-ORF7 and ORF7-GFP fusion proteins were expressed and the size of TGEV ORF7 was 9 ku. The analysis of combining the bioinformatics and confocal microscopy results showed that the TGEV ORF7 was located at the endoplasmic reticulum, and the subcellular localization might be induced by the N-terminal signal peptide. Furthermore, the real-time PCR results showed the viral RNAs were decreased by 99% in the cells with ORF7 protein over expression, indicating that TGEV ORF7 protein might regulate the replication of TGEV. These results provide some experimental reference for further revealing the function of ORF7 protein.