Discovery of Rotavirus NSP2 Regulation of Lipid Droplet Formation and an Unrecognized Pathophysiological Mechanism of Rotavirus-induced Diarrhea
| dc.contributor.advisor | Estes, Mary K | |
| dc.contributor.advisor | Crawford, Sue E | |
| dc.contributor.advisor | Stern, Michael | |
| dc.creator | Liu, zheng | |
| dc.date.accessioned | 2020-04-17T21:45:49Z | |
| dc.date.available | 2020-06-01T05:01:10Z | |
| dc.date.created | 2019-12 | |
| dc.date.issued | 2020-04-14 | |
| dc.date.submitted | December 2019 | |
| dc.date.updated | 2020-04-17T21:45:49Z | |
| dc.description.abstract | The overall goal of my thesis is to reveal the mechanism of RV-mediated LD formation, and the role RV exploitation of LD biogenesis plays in RV pathogenesis. Across all age groups, diarrhea is one of the top five causes of death world¬wide; while among children younger than 5 years old, diarrheal diseases are the second leading cause of morbidity and mortality around the world (Liu et al., 2012). Rotaviruses (RV), calici¬viruses (particularly norovirus), astroviruses, and enteric adenoviruses are the four predominant causes of viral gastroenteritis (Zachos, 2016). The symptoms of RV infection, such as acute watery diarrhea, nausea, vomiting and low-grade fever, can last for several days, and cause dehydration (Bishop, 2009). The molecular mechanisms of RV-induced gastroenteritis are not completely clear yet. Currently, there are three proposed mechanisms (1) osmotic diarrhea due to malabsorption, (2) secretory diarrhea due to the effects of the rotavirus nonstructural protein NSP4 expression, and (3) activation of the enteric nervous system (ENS) (Crawford et al., 2017). These mechanisms may be interrelated. RV replicates and assembles immature viral particles in cytoplasmic compartments named viroplasms. Viroplasms contains both viral and cellular components. Viroplasms associate with lipid droplets (LDs), neutral lipids and LD-specific proteins have been detected on viroplasms. Disrupting LD formation prevents viroplasm assembly and RV replication (Cheung et al., 2010). Diacylglycerol acyltransferase (DGAT1) is the key cellular enzyme in triacylglycerol synthesis required for LD biogenesis. Recent research reported that loss-of-function mutations in DGAT1 gene lead to severe congenital diarrheal disease in young children (Gluchowski et al., 2017; Haas et al., 2012; van Rijn et al., 2018). Patients with DGAT1 deficiency experience symptoms, such as non-bloody diarrhea, vomiting, and dehydration, similar to what is seen in RV-infected patients. My thesis research results (see chapter 3) demonstrate that DGAT1 is degraded in RV-infected cells by a proteasome-mediated mechanism involving ubiquitinated RV nonstructural protein NSP2. RV infection induced DGAT1-silencing results in earlier formation and an increased number of viroplasm/LDs per cell that translates into a 4-5 fold increase in viral yield (p<0.05). These results suggest that RV-induced DGAT1 degradation may trigger LD biogenesis and promote viroplasm/LD formation and lead to DGAT1 deficiency in the small intestine. DGAT1 deficiency is a rare cause of neonatal diarrhea purportedly due to the altered trafficking of key ion transporters to the apical brush border of enterocytes. Confocal microscopy results demonstrated that RV-mediated DGAT1 degradation results in a similar loss of the apical brush border transporters sucrase isomaltase and the sodium hydrogen exchanger protein as in DGAT1-/- HIEs. However, western blot analysis revealed that expression of these and other proteins, ezrin, villin, E-cadherin, and JAM-1, was substantially reduced, indicating an alternative mechanism for diarrhea. RV-mediated DGAT1 degradation or DGAT1 deficiency does not lead to global alterations in protein trafficking but instead results in loss of expression of proteins key to carbohydrate digestion, and water, electrolyte, and nutrient absorption. My results elucidate a new pathophysiological mechanism of RV-mediated and DGAT1 deficiency malabsorptive diarrhea. This research identified a previous unrevealed mechanism which contributes to the severe diarrheal disease induced by RV infection. This mechanism is not interrelated with the other proposed mechanisms, it is the first mechanism which recognizes the important roles of another viral protein (NSP2) rather than NSP4, and other cellular components (DGAT1) rather than Ca2+ signaling in RV-induced gastroenteritis. Since RV viroplasm requires LD biogenesis for particle assembly, additional research (see chapter 4) reports studies that explored the interaction between the viroplasm and LD-associated proteins. My results show that viroplasms are associated with a pool of LDs that are coated by PLIN1, but not PLIN2 or PLIN3. In addition, silencing PLIN1 by siRNA transfection or introducing homozygous mutation, significantly decreased the yield of RV in the cells by 50% to 90%, indicating the crucial role PLIN1 LDs may play in viroplasm formation. Co-Immunoprecipitation (co-IP) results show that PLIN1 physically interacts with both the viroplasm exclusive form of NSP2 (vNSP2) and the cytoplasmic dispersed form of NSP2 (dNSP2), whereas PLIN2 only interacts with dNSP2. In addition, more and larger PLIN1 coating LDs, which are also viroplasms, are observed in RV-infected cells. Almost no PLIN2 LD is observed in RV-infected cells, while many PLIN2 LDs can be seen in the neighboring uninfected cells. These results suggest PLIN2 may be degraded via its interaction with dNSP2 in a manner similar to how DGAT1 is degraded, whereas PLIN1 is stabilized in RV-infected cells. My western blot results show that the addition of MG132 inhibits the degradation of PLIN1 in mock- but not RV-infected cells, indicating RV infection inhibits PLIN1 turnover. Co-Immunoprecipitation results also show that similar amounts of both forms of NSP2 (d- and vNSP2) are pulled down by polyclonal PLIN1 antibody and phosphorylated specific monoclonal PLIN1 antibody, suggesting the phosphorylated PLIN1 is the isoform that interacts with both dNSP2 and vNSP2. Phosphorylated PLIN1 is reported to be exclusively located on LDs rather than other cellular components, such as the endoplasmic reticulum, peroxisomes, and mitochondria localized near LDs (Blanchette-Mackie et al., 1995). The interactions between phosphorylated PLIN1 and both d- and vNSP2 indicate viroplasm components are recruited to the surface of PLIN1 LDs, suggesting a scaffolding role of PLIN1 during viroplasm formation. Together, my research shows RV infection may reshape cellular lipid metabolism and other cellular pathways via the physical interaction between the two forms of the RV NSP2 protein and multiple cellular targets. Interaction with dNSP2 alone, which is K48- ubiquitinated and interacts with multiple units of the 26S proteasome, may promote the proteasome-mediated degradation of a cellular protein and the interaction with vNSP2 may stabilize or sequester the cellular proteins to viroplasm. We propose this is a previously unrecognized mechanism of RV-induced pathogenesis. | |
| dc.embargo.terms | 2020-06-01 | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.citation | Liu, zheng. "Discovery of Rotavirus NSP2 Regulation of Lipid Droplet Formation and an Unrecognized Pathophysiological Mechanism of Rotavirus-induced Diarrhea." (2020) Diss., Rice University. <a href="https://hdl.handle.net/1911/108302">https://hdl.handle.net/1911/108302</a>. | |
| dc.identifier.uri | https://hdl.handle.net/1911/108302 | |
| dc.language.iso | eng | |
| dc.rights | Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder. | |
| dc.subject | Rotavirus | |
| dc.subject | lipid droplet | |
| dc.subject | NSP2 | |
| dc.subject | DGAT1 | |
| dc.subject | PLIN1 | |
| dc.title | Discovery of Rotavirus NSP2 Regulation of Lipid Droplet Formation and an Unrecognized Pathophysiological Mechanism of Rotavirus-induced Diarrhea | |
| dc.type | Thesis | |
| dc.type.material | Text | |
| thesis.degree.department | Biochemistry and Cell Biology | |
| thesis.degree.discipline | Natural Sciences | |
| thesis.degree.grantor | Rice University | |
| thesis.degree.level | Doctoral | |
| thesis.degree.major | Virology | |
| thesis.degree.name | Doctor of Philosophy |
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