Niemann-pick C1 is essential for ebolavirus replication and pathogenesis in vivo.

UNLABELLED: Recent work demonstrated that the Niemann-Pick C1 (NPC1) protein is an essential entry receptor for filoviruses. While previous studies focused on filovirus entry requirements of NPC1 in vitro, its roles in filovirus replication and pathogenesis in vivo remain unclear. Here, we evaluated the importance of NPC1, and its partner in cholesterol transport, NPC2, by using a mouse model of Ebolavirus (EBOV) disease. We found that, whereas wild-type mice had high viral loads and succumbed to EBOV infection, Npc1(-/-) mice were entirely free of viral replication and completely protected from EBOV disease. Interestingly, Npc1(+/-) mice transiently developed high levels of viremia, but were nevertheless substantially protected from EBOV challenge. We also found Npc2(-/-) mice to be fully susceptible to EBOV infection, while Npc1(-/-) mice treated to deplete stored lysosomal cholesterol remained completely resistant to EBOV infection. These results provide mechanistic evidence that NPC1 is directly required for EBOV infection in vivo, with little or no role for NPC1/NPC2-dependent cholesterol transport. Finally, we assessed the in vivo antiviral efficacies of three compounds known to inhibit NPC1 function or NPC1-glycoprotein binding in vitro. Two compounds reduced viral titers in vivo and provided a modest, albeit not statistically significant, degree of protection. Taken together, our results show that NPC1 is critical for replication and pathogenesis in animals and is a bona fide target for development of antifilovirus therapeutics. Additionally, our findings with Npc1(+/-) mice raise the possibility that individuals heterozygous for NPC1 may have a survival advantage in the face of EBOV infection. IMPORTANCE: Researchers have been searching for an essential filovirus receptor for decades, and numerous candidate receptors have been proposed. However, none of the proposed candidate receptors has proven essential in all in vitro scenarios, nor have they proven essential when evaluated using animal models. In this report, we provide the first example of a knockout mouse that is completely refractory to EBOV infection, replication, and disease. The findings detailed here provide the first critical in vivo data illustrating the absolute requirement of NPC1 for filovirus infection in mice. Our work establishes NPC1 as a legitimate target for the development of anti-EBOV therapeutics. However, the limited success of available NPC1 inhibitors to protect mice from EBOV challenge highlights the need for new molecules or approaches to target NPC1 in vivo.

Inquiry-based learning: inflammation as a model to teach molecular techniques for assessing gene expression.

This laboratory module simulates the process used by working scientists to ask and answer a question of biological interest. Instructors facilitate acquisition of knowledge using a comprehensive, inquiry-based approach in which students learn theory, hypothesis development, experimental design, and data interpretation and presentation. Using inflammation in macrophages as a model system, students perform a series of molecular biology techniques to address the biological question: "Does stimulus 'X' induce inflammation?" To ask this question, macrophage cells are treated with putative inflammatory mediators and then assayed for evidence of inflammatory response. Students become familiar with their assigned mediator and the relationship between their mediator and inflammation by conducting literature searches, then using this information to generate hypotheses which address the effect of their mediator on induction of inflammation. The cellular and molecular approaches used to test their hypotheses include transfection and luciferase reporter assay, immunoblot, fluorescence microscopy, enzyme-linked immunosorbent assay, and quantitative PCR. Quantitative and qualitative reasoning skills are developed through data analysis and demonstrated by successful completion of post-lab worksheets and the generation and oral presentation of a scientific poster. Learning objective assessment relies on four instruments: pre-lab quizzes, post-lab worksheets, poster presentation, and posttest. Within three cohorts (n = 85) more than 95% of our students successfully achieved the learning objectives.

CTP:phosphocholine cytidylyltransferase alpha is required for B-cell proliferation and class switch recombination.

CTP:phosphocholine cytidylyltransferase (CCT) is a key rate-controlling enzyme in the biosynthetic pathway leading to the principle membrane phospholipid, phosphatidylcholine. CCTalpha is the predominant isoform expressed in mammalian cells. To investigate the role of CCTalpha in the development and function of B-lymphocytes, mice with B-lymphocytes that selectively lacked CCTalpha were derived using the CD19-driven Cre/loxP system. When challenged with a T-cell-dependent antigen, the animals harboring CCTalpha-deficient B-cells exhibited a hyper-IgM secretion phenotype coupled with a lack of IgG production. The inability of CCTalpha-/- B-cells to undergo class switch recombination correlated with a proliferation defect in vivo and in vitro in response to antigenic and mitogenic stimuli. Lipopolysaccharide stimulation of CCTalpha-/- B-cells resulted in an early trigger of the unfolded protein response-mediated splicing of Xbp-1 mRNA, and this was accompanied by accelerated kinetics of IgM secretion and higher incidence of IgM-secreting cells. Thus, the inability of stimulated B-cells to produce enough phosphatidylcholine prevents proliferation and class switch recombination but leads to unfolded protein response activation and a hyper-IgM secretion phenotype.

Evidence that marginal zone B cells possess an enhanced secretory apparatus and exhibit superior secretory activity.

Marginal zone B (MZB) cells are the first splenic B cells to initiate Ab secretion against polysaccharide-encapsulated Ags in vivo. This swift MZB cell response can be reproduced in vitro as LPS treatment induces Ab secretion in as little as 12 h. Conversely, in vitro LPS treatment of splenic follicular B (FOB) cells results in Ab secretion after 2-3 days. The basis for these distinct response kinetics is not understood. We performed ex vivo analysis of resting and LPS-stimulated murine MZB and FOB cells and found that MZB cells express higher levels of the LPS TLR complex RP105/MD-1 and respond to much lower concentrations of LPS than do FOB cells. Furthermore, increasing doses of LPS do not accelerate the kinetics by which FOB cells transition into Ab secretion. Ultrastructural analysis of resting cells demonstrated that rough endoplasmic reticulum is more abundant in MZB cells than in FOB cells. Additionally, RT-PCR and immunoblot analyses revealed that numerous endoplasmic reticulum resident chaperones and folding enzymes are expressed at greater levels in resting MZB cells than in resting FOB cells. Although both LPS-stimulated MZB and FOB cells increase expression of these factors, MZB cells exhibit a more rapid increase that correlates with accelerated kinetics of Ab secretion and higher per cell output of secreted IgM. These data indicate that MZB cells are equipped for exquisite sensitivity to bacterial components like LPS and poised for rapid, robust Ab production, making MZB cells ideally suited as frontline defenders in humoral immunity.

A role for the unfolded protein response in optimizing antibody secretion.

Terminal differentiation of B lymphocytes into antibody(Ab)-secreting plasma cells is marked by a sharp rise in immunoglobulin (Ig) biosynthesis that increases demand on the protein folding capacity of the endoplasmic reticulum (ER). The unfolded protein response pathway (UPR) allows cells to respond to challenging conditions within the ER, in part by the activities of the XBP1 and ATF6alpha/beta transcription factors. The UPR is activated in differentiating B cells, and XBP1 is required for the generation of Ab-secreting plasma cells. Therefore, it has been hypothesized that the UPR mediates ER homeostasis as B cells transition into high-rate Ab secretion. We sought to test this hypothesis in primary murine splenic B cells stimulated to secrete Ab in vitro. Here, we report that enforced expression of a dominant-negative ATF6alpha mutant in differentiating B cells reduces the output of secreted IgM and increases improper release of IgM assembly intermediates. These data indicate that the UPR functions to optimize the efficiency of Ab secretion and provide new insight into the fundamental role of the UPR in humoral immunity.

Stressed-out B cells? Plasma-cell differentiation and the unfolded protein response.

Plasma cells operate as factories where large quantities of Ig heavy and light chains are made and assembled into functional antibodies. The finished products are shipped out with impressive efficiency. A major component of the machinery necessary for high-rate antibody secretion is an elaborate network of endoplasmic reticulum (ER), the site of antibody biosynthesis. Recent discoveries have provided insights into how this expansive secretory machinery is built, equipped and maintained. The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the ER, regulates the expression and activity of X-box binding protein 1, a transcription factor required for plasma-cell development. The UPR pathway therefore senses conditions in the ER--the very compartment where antibodies are formed--and directs events required for humoral immunity.

Modulation of CRP-dependent transcription at the Escherichia coli acsP2 promoter by nucleoprotein complexes: anti-activation by the nucleoid proteins FIS and IHF.

acs encodes acetyl-coenzyme A synthetase, a high-affinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acsP2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP. Using in vitro transcription assays, we demonstrated that FIS and IHF independently reduce CRP-dependent acs transcription. Using in vivo reporter assays, we showed that disruption of DNA sites for FIS or deletion of DNA sites for IHF increases acs transcription. We propose that FIS and IHF each function directly as anti-activators of CRP, each working independently at different times during growth to set the levels of CRP-dependent acs transcription.