Our long-term goal is to understand how rotavirus (RV) exploits cellular pathways such as autophagy membranes and lipid droplet (LD) formation to their benefit. RVs remain significant human pathogens in spite of the introduction of vaccines. RVs are outstanding models to understand the fundamental molecular biology of cell signaling as RVs utilize intracellular calcium ([Ca2+]i), and co-opt intracellular membranes, and autophagy, important cellular pathways manipulated by many viruses. Recently cellular LDs were found to be components of de novo synthesized cytoplasmic organelles (viroplasms) in RV-infected cells. Viroplasms provide a physical platform for efficient viral replication and maturation. LDs are dynamic, multi-functional intracellular organelles involved in lipid storage and metabolism, and they play essential roles in several viral and intracellular bacterial infections. LDs are also important in many aspects of health and disease (metabolism, diabetes, obesity, heart disease). However, fundamental information on the biology and function of LDs and infectious processes remains limited, and the relationships between RV replication, in particular, and LD components are poorly understood. The biogenesis, growth and maturation of LDs and viroplasms appear to share many similarities. Are viroplasms modified LDs? Our proposed studies on LDs and viroplasms build on our recent work that discovered how RV induces changes in Ca2+ homeostasis critical for RV replication, and how cellular autophagy affects RV replication. We made several exciting discoveries including finding cellular autophagy is required for RV replication and viroplasm assembly, and autophagy is initiated by a RV viroporin that increases cytoplasmic Ca2+ and activates Ca2+ signaling pathways; this has multiple downstream effects including initiation of autophagy by activation of a CAMKK2 and AMPK-dependent signaling pathway. RV subsequently suppresses autophagy maturation, and hijacks the membrane trafficking function of autophagy to transport ER-associated viral glycoproteins to mature viroplasms for viral morphogenesis. Finally, viroplasm formation requires LD formation, which may be regulated by the viroporin and autophagy proteins. We hypothesize that RV infection induces LDs, affects the composition of LDs and usurps LD components to initiate viroplasm formation and support viroplasm maturation to coordinate RNA replication and initial particle assembly. We propose experiments to answer two questions. (1) What is the molecular basis of initial lipid droplet formation and growth in RV-infected cells? (2) What molecular mechanisms regulate viroplasm initiation, growth and maturation? These studies are significant and of fundamental interest 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.
Rotaviruses (RVs) remain major pathogens that cause life-threatening diarrheal disease in children under 5 years of age and result in nearly half a million deaths annually. Although RV vaccines are available, they do not work optimally in countries where they are needed most, and the emergence of new virus strains in vaccinated individuals is raising questions about whether vaccine efficacy will remain high. Our proposed studies are designed to understand how RVs takeover and use key host cell physiologic pathways involved in fat (lipid) and intracellular membrane remodeling to enhance their own 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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| 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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