Primary antibodies were purchased as follows: anti-HA (Abcam), anti-myc (EMD Millipore), anti-BiP (Santa Cruz Biotechnology), anti-CHOP (Santa Cruz Biotechnology), and anti–actin (Sigma-Aldrich). in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell typeC or prion Rabbit Polyclonal to mGluR4 strainCspecific. Moreover, prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy. and models that prion infection resulted in cells undergoing ER stress, which further facilitates the formation of misfolded PrPC and increased prion conversion (22, 24,C26). Previous studies in our laboratory have also Evocalcet demonstrated a Evocalcet direct influence of impairment in quality control mechanisms on prion conversion, and overexpression of quality control proteins such as ERGIC-53 and EDEM-3 reduced prion conversion (24). Another group showed that overexpression of BiP modulated prion propagation and in animal models (27). Thus, the manipulation of cellular quality control mechanisms could be a potential strategy for interfering in prion conversion by helping only correctly folded PrPC to reach the plasma membrane, which is less prone to prion conversion. Additionally, Evocalcet it has been reported that ERp57 has a protective effect against prion toxicity and regulates the expression and maturation of PrPC in cells (28, 29). In this study, we investigated the role of overexpression of proteins involved in folding (ERp57) and secretory protein cargo transport (VIP36) on prion conversion. In persistently prion-infected cells, we found a significant reduction of PrPSc following overexpression. We used both stable and transient overexpression systems, different cell types, and different prion strains to Evocalcet assess the effect on prion propagation. Moreover, when ERp57- or VIP36-overexpressing noninfected cells were infected with prions, we found that the overexpressing cells were less susceptible to prion infection. Additionally, ERp57-overexpressing cells showed reduced susceptibility to induction of ER stress. These results provide strong evidence for the role of quality control in prion infection. Together with our preliminary data, this suggests that ERp57 and VIP36 could be promising targets against prion infection. Thus, manipulation of the protein quality control mechanisms could lead to reduced PrPSc conversion. Results Stable overexpression of ERp57 or VIP36 reduces PrPSc in prion-infected neuroblastoma cells To investigate the role of ERp57 and VIP36 in prion replication, we stably overexpressed ERp57 or VIP36 in N2a cells persistently infected with mouse-adapted scrapie prion strain 22L (ScN2a-22L) Evocalcet using a lentiviral gene integration technique. ScN2a-22L cells were transduced with lentiviruses that integrated genes encoding ERp57 (HA-tagged) or VIP36 (myc-tagged) into the host genome, allowing stable overexpression of genes. Transduced cells were selected using puromycin. When nonvirally transduced cells were subjected to puromycin selection as a control, all cells were susceptible to puromycin treatment. As lentiviral transduction resulted in expression of GFP along with the target gene (dual promoter construct), successful transduction and selection of cells were confirmed by investigating GFP autofluorescence with fluorescence microscopy and target protein expression with Western blotting. The transduced cells were passaged. At each passage, cells were lysed, and the lysates were subjected to PK digestion and immunoblotting. Upon overexpression of ERp57, we found a significant reduction of PrPSc in the first passage compared with control cells transduced with mock virus (Fig. 1, and = 5C8). **, 0.01; ***, 0.001. Moreover, we tested cells for changes in prion seeding activity using real-time quaking-induced conversion (RT-QuIC) assay. In this test, recombinant PrPC substrate is converted into ThT-binding aggregates in.