The ORF6 protein exerts an effect on the expression of transgenes from co-transfected expression constructs
To determine amount of ORF6 required to exert an effect on the expression of transgenes from co-transfected expression constructs, Vero E6 cells were transfected with 1 μg of pXJmyc-nsp8 alone or with either 1 μg, 2 μg or 3 μg of pXJ3'-ORF6 plasmid. Western blotting showed a dose-dependent reduction in the expression of nsp8, concomitant with an increase in the expression of ORF6 (Figure 1A). In order to determine the specificity of this effect, Vero E6 cells were cotransfected with another mammalian expression construct pXJflag-GST and pXJ3'-ORF6 in the same manner as previously done with the construct encoding for nsp8. When titrated against increasing amounts of ORF6 protein, the flag-tagged GST protein was also observed by Western Blotting to show reduced levels of expression in a dose-dependent manner (Figure 1B). Thus, it seems that ORF6 exerts an effect on the expression of transgenes from co-transfected expression constructs regardless of the nature of the transgenes. However, when increasing amounts of GST were titrated against nsp8 in the same manner, no significant reduction in nsp8 expression was observed, indicating that this effect was specific to the ORF6 protein (Figure 1C).
ORF6 does not affect total cellular protein synthesis
The ORF6 protein has been observed to cause a reduction in the expression of 2 different proteins, with different epitope tags. In order to examine the possibility that this downregulation could be a global effect, Vero E6 cells were transfected with either empty vector, pXJ3'HA, or pXJ3'-ORF6, and metabolically labeled with 35S for a period of 30 minutes. Quantification of the signals in each lane of the resulting autoradiograph was performed and the readings were normalized to the reading in the lane from the untransfected cells (Figure 2). When 1 μg of DNA was used, there is a slight decrease in the total cellular protein synthesis in ORF6 expressing cells when compared to cells which had been transfected with empty vector. However, almost the same degrees of decrease in the total cellular protein synthesis were observed in both pXJ3'HA and pXJ3'-ORF6 transfected cells when 2 μg of DNA was used. Hence, the decrease in total cellular protein synthesis observed seems to be related to the transfection process rather than the expression of ORF6. This suggests that the cellular effect of ORF6 is not global.
ORF6 exerts its effect at a transcriptional level
In order to examine the possibility that ORF6 affects the expression of transgenes from co-transfected expression constructs via a transcriptional mechanism, Vero E6 cells were transfected with 1 μg of pXJ40myc-nsp8 and either 1 μg, 2 μg of pXJ3'-ORF6 or no ORF6 plasmid. 16 hours post-transfection, total RNA was extracted, reverse transcribed and subjected to quantitative real-time PCR. Taqman chemistry was used to assay for nsp8 and GAPDH was used as an endogenous control. The ΔΔCt method was used to calculate the nsp8 mRNA level with respect to the level in the absence of ORF6. As shown in Figure 3, ORF6 caused a reduction in the level of nsp8 mRNA in a dose-dependent manner, indicating a transcriptional reduction caused by ORF6. These results suggest that ORF6 is able to exert some form of transcriptional inhibition, which is seen in the reduced expression from co-transfected plasmids.
The ORF6 protein localizes to intracellular vesicles positive for Lamp1 and CD63
It has been previously reported that the SARS-CoV ORF6 protein localizes to intracellular membranous compartments, which have been suggested to be induced by ORF6 itself (10). In agreement, it was observed in this study that ORF6 localized to vesicular compartments both in SARS-infected Vero E6 cells and Vero E6 cells transfected with a plasmid encoding for the ORF6 protein (Figure 4A). Vero E6 cells were infected with SARS-CoV and analyzed by immunofluoresence, using an antibody against the ORF6 protein. ORF6 was observed to localize to a distinct population of intracellular vesicles in these infected cells (Figure 4A). Following this, a mammalian expression plasmid pXJ3'-ORF6 was transfected into Vero E6 cells and analyzed in the same manner with the same antibody. Confocal microscopy showed that the ORF6 protein localized to a similar population of intracellular vesicles.
In our previous work studying the interaction between ORF6 and the nsp8 protein of SARS-CoV (5), we showed a colocalization of ORF6 and Lamp1 in Vero E6 cells infected with the HKU39849 strain of SARS-CoV. Similarly, there was a significant degree of colocalization between the Lamp1 and ORF6 in transiently transfected Vero E6 cells (Figure 4B), indicating that the ORF6-positive vesicles seen in both infected cells and transiently transfected Vero E6 cells were probably an identical population of vesicles. This allowed us to then use pXJ3'-ORF6-transfected Vero E6 cells to further study the characteristics of this vesicular population. In addition to Lamp1, CD63 (a marker for late endosomes) was used to examine the compartmental characteristics of the vesicles. As shown in Figure 4B, ORF6-positive vesicles coincide significantly with CD63-positive vesicles in Vero E6 cells. This population of vesicles is therefore a subset of the late endosomal and lysosomal populations, and shows that the plasmid system employed here yields similar colocalization of ORF6 with cellular markers as seen in previous infection work (5).
Amino Acids 53-56 in ORF6 constitute a putative diacidic motif which affects the suppression of the expression of co-transfected myc-nsp8
Using the CBS Prediction Servers (http://www.cbs.dtu.dk/services/), it was determined that the ORF6 protein had several putative motifs of interest. Of these, aa49-52 (YSEL) was predicted to be a lysosomal targeting motif YXXL (18), and aa53-56 (DDEE) bears similarity to a putative diacidic motif DxE, which governs ER export and subsequent localization to different membranous compartments (19). These motifs were also predicted by Netland and co-workers (4). In order to determine the contribution of these motifs to the function of the ORF6 protein, alanine substitutions were introduced by two-step PCR to yield ORF6A49-52, which substituted four alanine residues for the YSEL region, and ORF6A53-56, which substituted four alanines for the DDEE region (Figure 5A).
These alanine substitution mutants were cloned into the same vector as the wildtype ORF6 gene and titrated against the nsp8 gene, by co-transfection of Vero E6 cells with plasmids encoding for myc-nsp8 and either wildtype ORF6, ORF6A49-52 or ORF6A53-56. It was observed that both mutants showed increased levels of expression compared to wildtype ORF6, and this observation was reproducible (data not shown). The effect of ORF6A49-52 mutant on the expression of the nsp8 gene was similar to wildtype ORF6 (Figure 5B). On the other hand, the nsp8 expression in the presence of ORF6A53-56 was higher than that in the presence of wildtype ORF6 (Figure 5B), which suggest that ORF6A53-56 is less efficient in suppressing the expression of co-transfected myc-nsp8.
As the difference between wildtype ORF6 and ORF6A53-56 was subtle, a more quantitative approach to assay nsp8 expression was deemed necessary. Hence, Vero E6 cells were transfected in the same manner and instead RNA was extracted and reverse transcribed and subjected to SYBR Green QPCR using primers for nsp8. GAPDH was used as a housekeeping gene in order to normalize the expression levels seen. When pXJmyc-nsp8 was co-transfected with 1 μg of pXJ3'-ORF6, ORF6A49-52 or ORF6A53-56, the levels of nsp8 mRNA were similar (Figure 5C). However, at 2 μg, the levels of nsp8 mRNA for both ORF6 mutants were slightly higher than wild-type ORF6 (Figure 5C). In order to determine if these differences are statistically significant, 5 independent experiments were performed, and the results were then used to perform a t-test measuring the significance of the difference between nsp8 expression levels when either 2 μg of wildtype ORF6 or 2 μg of each mutant were used. The difference in nsp8 expression when co-transfected with either wildtype ORF6 and ORF6A49-52 is not statistically significant (a two-tailed p-value of 0.08). On the other hand, the difference in nsp8 expression when co-transfected with either wildtype ORF6 and ORF6A53-56 is statistically significant (a two-tailed p-value of 0.01). This indicated that the reduction in the suppression of the expression of co-transfected myc-nsp8 by ORF6A53-56 was significant, and therefore that the putative diacidic motif defined by amino acids 53-56 has a role to play in this ability of the ORF6 protein. The ORF6A49-52 mutant did not show as high a level of significance.
Alanine substitutions at amino acids 53-56 alter the subcellular localization of ORF6
Next, the subcellular localization of ORF6A49-52 and ORF6A53-56 was compared to wildtype ORF6. Vero E6 cells were transiently transfected with either pXJ3'-ORF6, pXJ3'-ORF6A49-52 or pXJ3'-ORF6A53-56 and subjected to immunofluoresence analysis. It was observed that the localization of ORF6A49-52 was somewhat similar to wild-type ORF6 with main expression in distinctive vesicles (Figure 6). However, slightly more ORF6A49-52 than wildtype ORF6 was found to spread diffusely throughout the cytoplasm. The ORF6A53-56 also has some cytoplasmic staining but it was also observed that this mutant protein localized to vesicles that were clustered into groups of 3-5 vesicles to form large aggregates. Careful examination of the localization of the wildtype protein did not yield similar observations of clusters. This indicated that the putative diacidic motif from amino acids 53-56, in addition to being involved in the suppression of the expression of co-transfected myc-nsp8, is also involved in the subcellular localization of the ORF6 protein and therefore these 2 phenomena may be linked.