Our long-term goal is to understand how rotavirus (RV) exploits cellular pathways such as autophagy membranes, calcium homeostasis, and lipid droplet (LD) formation to enhance their replication and cause disease. RVs remain significant human pathogens in spite of the introduction of vaccines. Several aspects of RV replication are unique yet broadly relevant to other viruses, such as cytoplasmic organelles (viroplasms, VI) formed by both viral and cellular proteins and LDs that form a physical platform for efficient viral replication and maturation. LDs are dynamic, multi-functional intracellular organelles involved in lipid storage and metabolism as well as in signal transduction, membrane trafficking and modulation of immune and inflammatory responses. LDs play essential roles in several viral and intracellular bacterial infections and are important in many aspects of health and disease (metabolism, diabetes, obesity, heart disease). However, mechanistic information of the interplay between lipid accumulation and these pathogens, and disease is far from complete. Our proposed studies on LDs and VIs build on our recent work. While the viral proteins NSP2 and NSP5 are known to be required for VI formation, the molecular mechanisms of how these two proteins associate with each other as well as with other viral and cellular proteins and LD components to form VI/LDs remain to be elucidated. We discovered two forms of NSP2 that interact with different isoforms of NSP5: a dispersed (dNSP2) form interacts with hypo-phosphorylated NSP5 and a previously recognized VI (vNSP2) form interacts with hyper- phosphorylated NSP5. We elucidated a novel phosphorylation-dependent mechanism for VI formation, in which the ubiquitous, constitutively active cellular protein kinase CK1? partially controls the assembly of RV VIs by phosphorylating NSP2 to trigger NSP2 octamer-octamer lattice formation. We also discovered that NSP2 is an autokinase and predict that NSP2 may phosphorylate other viral or cellular proteins for VI assembly and RV replication. We hypothesize that interactions of RV and cellular proteins in specialized microdomains of the endoplasmic reticulum nucleate and induce VI/LDs essential for virus replication, affect the composition of the LD-associated proteins and result in previously unrecognized mechanisms of RV-induced pathogenesis. We propose experiments to answer three questions. (1) How do NSP4, NSP2 and specialized microdomains in the ER lead to nucleation of VI/LDs? (2) How does phosphorylation orchestrate VI formation and the conversion of dNSP2 to vNSP2 to initiate VI/LD formation and subsequent VI/LD maturation? (3) How does DGAT1 degradation lead to LD formation and what are the specific roles of PLIN1 and PLIN3 LDs in RV infection and pathogenesis? These studies are significant because viral perturbations of host signaling and metabolic pathways that involve LDs are critical for multiple pathogens. Because RVs replicate in enterocytes in the small intestine, the major site of fat absorption in the body, understanding the effects of RV infection on LD biology has the potential to reveal new insights into the consequences of virus infection on host metabolism.
Rotavirus (RV) infections kill an estimated 130,000-215,000 young children globally in spite of the recent introduction of vaccines, highlighting the importance for continued study of this pathogen. Our proposed studies are designed to understand how RVs take advantage of key host cellular pathways involved in the accumulation of fat (lipid) and cellular membranes to form specialized sites in the infected cell critical for virus replication. We expect the knowledge gained from our studies will help (1) understand how RVs and many other viruses cause disease, (2) explain why RV infections in children become life-threatening, and (3) lead to new treatment and antiviral therapies.
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| Hu, Liya; Sankaran, Banumathi; Laucirica, Daniel R et al. (2018) Glycan recognition in globally dominant human rotaviruses. Nat Commun 9:2631 |
| Blutt, Sarah E; Crawford, Sue E; Ramani, Sasirekha et al. (2018) Engineered Human Gastrointestinal Cultures to Study the Microbiome and Infectious Diseases. Cell Mol Gastroenterol Hepatol 5:241-251 |
| Ramani, Sasirekha; Crawford, Sue E; Blutt, Sarah E et al. (2018) Human organoid cultures: transformative new tools for human virus studies. Curr Opin Virol 29:79-86 |
| Bányai, Krisztián; Estes, Mary K; Martella, Vito et al. (2018) Viral gastroenteritis. Lancet 392:175-186 |
| Shanker, Sreejesh; Hu, Liya; Ramani, Sasirekha et al. (2017) Structural features of glycan recognition among viral pathogens. Curr Opin Struct Biol 44:211-218 |
| Zou, Winnie Y; Blutt, Sarah E; Crawford, Sue E et al. (2017) Human Intestinal Enteroids: New Models to Study Gastrointestinal Virus Infections. Methods Mol Biol : |
| Blutt, Sarah E; Broughman, James R; Zou, Winnie et al. (2017) Gastrointestinal microphysiological systems. Exp Biol Med (Maywood) 242:1633-1642 |
| Crawford, Sue E; Ramani, Sasirekha; Tate, Jacqueline E et al. (2017) Rotavirus infection. Nat Rev Dis Primers 3:17083 |
| Vernetti, Lawrence; Gough, Albert; Baetz, Nicholas et al. (2017) Functional Coupling of Human Microphysiology Systems: Intestine, Liver, Kidney Proximal Tubule, Blood-Brain Barrier and Skeletal Muscle. Sci Rep 7:42296 |
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