This represents a novel approach to antagonize ER/Golgi trafficking, as no other cellular or microbial protein has been described to use a motif similar to an ER export signal to gain access to and antagonize the secretory pathway. This Benzoylaconine vector encodes the gene of interest under a CMV promoter and SEAP, a reporter protein that is rapidly secreted from cells and is a quantitative surrogate of protein secretion, which is translated via an internal ribosomal sequence. All proteins expressed using this vector system had N terminal GFP tags, which has been reported to not affect the ability of PV 3A to inhibit protein secretion. At various times post-transfection, media and cell pellets were assayed for extra and intra-cellular enzymatic SEAP activity, respectively; total SEAP levels did not significantly differ between all proteins expressed at any time point tested. For all constructs, enzymatic SEAP activity was first detectable over background in both fractions at 6 hpt. Expression of GFP alone led to,75% secreted SEAP throughout the assay, whereas expression of PV 3A led to a significant reduction of SEAP secretion with maximal reduction to 38%, or 53% of GFP alone levels, at the final time point, which is similar to previous results. With similar kinetics to 3A, NV p22 also ultimately inhibited SEAP secretion to 34%, or 48% of GFP alone levels. From this, we concluded that, despite their differing specific effects on Golgi phenotype, NV p22 is able to inhibit SEAP secretion, and therefore cellular protein secretion, to levels similar to PV 3A. To gain a better understanding of potential ultrastructural alterations induced by p22, cells expressing GFP or GFP-p22 were flow sorted for GFP expression at 24 hpt. After 24 hours of recovery following flow sorting, and therefore 48 hpt, cells were fixed and thin sections were visualized by electron microscopy. After flow sorting, cells expressing GFP alone had intact and peri-nuclearly localized Golgi with cisternal stacks clearly visible in 31 of 59 cells examined. In contrast, cells expressing GFP-p22 had detectable Golgi stacks in just 4 of 57 cells examined. Instead, GFP-p22 cells exhibited an Lomitapide Mesylate abundance of large vacuoles, loose single membranes, and double-membrane structures. Many of these structures had what appeared to be cargo inside them, but the nature of this cargo was unclear as these structures were much larger than would be expected for those containing normal secretory pathway cargo. These results confirmed the immunofluorescence observations of a disassembled and phenotypically abnormal Golgi, demonstrating that expression of p22 led to rearrangements and alterations to various components of the secretory pathway, as would be expected during antagonism of this pathway. To explore if this conserved norovirus motif could play a role in the previously demonstrated inhibition of cellular protein secretion by p22, individual alanine mutations were made within each of the conserved residues within the putative ER export signal of p22 and tested as before using the SEAP system. Mutations within the S, D and G residues had no effect on the ability of p22 to inhibit cellular protein secretion; however, mutation of both the Y and E residues led to intermediate levels of protein secretion. When these two residues were combined into a single AXWASDG construct, SEAP secretion at 36 hpt was not statistically different from that of GFP alone. Total SEAP expressed by all p22 mutants did not significantly differ from that of wildtype p22 and similar levels of all p22 proteins were expressed, as confirmed by western blot analysis of intracellular fractions at 36 hpt, showing that the 
observed decreases in inhibition of protein secretion were not due to changes in protein expression or stability. To examine Golgi phenotype in cells expressing the AXWASDG mutant of p22, we next explored the phenotype of the Golgi in cells expressing this construct. By electron microscopy at 48 hpt and after flow sorting, cells expressing p22 had wildtype Golgi with intact cisternae present in 26 of 56 cells.