A putative diacidic motif in the SARS-CoV ORF6 protein influences its subcellular localization and suppression of expression of co-transfected expression constructs

Gunalan, Vithiagaran; Mirazimi, Ali; Tan, Yee-Joo

BACKGROUND: The ORF6 protein is one of the eight accessory proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV). Numerous properties of ORF6 have been documented and this study focuses on two of these, namely, its ability to suppress the expression of co-transfected expression constructs and its subcellular localization to vesicular structures. RESULTS: Using a transient transfection system, ORF6's ability to suppress the expression of co-transfected expression constructs was measured in a quantitative manner. While ORF6 does not have a global effect on protein synthesis, quantitative real-time PCR revealed that it down-regulated the mRNA level of the co-transfected myc-nsp8 gene. Furthermore, alanine substitution of a diacidic cluster motif (aa53-56) in the ORF6 gene caused a reduction in the suppression of expression of co-transfected myc-nsp8 gene. Our previous study revealed that ORF6 localized to vesicular structures in SARS-CoV infected Vero E6 cells. Here, ORF6 was observed to be localized to similar vesicular structures in Vero E6 cells which have been transiently transfected with a mammalian expression plasmid encoding for untagged ORF6. ORF6 showed partial colocalization with cellular proteins CD63 and Lamp1, suggesting that the vesicular structures may be a subpopulation of endosomal/lysosomal vesicles. The alanine substitution of the diacidic cluster motif also altered the subcellular localization of the ORF6 protein, indicating a potential relationship between the subcellular localization of the ORF6 protein and its ability to suppress the expression of co-transfected expression constructs. CONCLUSIONS: By combining quantitative real-time PCR and transient transfection system, a simple and safe method is established to measure ORF6's ability to suppress the expression of co-transfected myc-nsp8. In addition, immunofluorescence analysis revealed that the subcellular localization of ORF6 when expressed on its own is similar to that observed in SARS-CoV infected cells. Through the use of these two assays, a putative diacidic motif in the ORF6 protein was found to influence its subcellular localization and ability to suppress the expression of co-transfected expression constructs.