Investigating the role of viroplasm formation and calcium levels on the production of prostaglandin E₂ during rotavirus infection
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Date
2022
Authors
Sander, Willem Jacobus
Journal Title
Journal ISSN
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Publisher
University of the Free State
Abstract
Both in vitro and in vivo studies have shown that levels of prostaglandin E₂ (PGE₂), an immunomodulatory eicosanoid, are increased during rotavirus (RV) infection. Although it has been shown that inhibition of cyclooxygenase (COX) (PGE₂ biomes) has adverse effects on viral yield, the mechanism of PGE₂ induction during replication remains unknown. Viroplasms are viral factories that consist of several viral proteins, in particular NSP2 and NSP5, and cellular lipid droplets. Lipid droplets (LDs), with their high content of neutral lipids and the proximity of PGE₂ biosynthetic enzymes, are well known sites for PGE₂ biosynthesis. In addition, during replication, RV has been shown to increase the total lipid content of infected cells, while modulating specific lipid classes during infection. Inhibitors that prevent the formations of LDs severely limit the amount of viroplasms formed and subsequently decrease viral progeny production. Another viral protein critical for viral replication is the enterotoxin, NSP4. NSP4, contains a viroporin domain that selectively conducts calcium (Ca²⁺) from the endoplasmic reticulum to the cytosol, increasing free intracellular Ca²⁺. Intracellular Ca²⁺ levels are crucial for the activation and function of cytoplasmic phospholipase A₂, the rate-limiting enzyme in PGE₂ biosynthesis. Both LDs and phospholipase A₂ are also essential for PGE₂ biosynthesis. Therefore, the main objective of the study was to determine when and by which mechanism(s) RV induces/amplifies the production of prostaglandin E₂. During early infection, RV attaches to several cellular receptors and enters the cells by either clathrin-dependent or -independent endocytosis. Other viruses such as bovine ephemeral fever virus haven been shown to require the activation COX-2-mediated PGE₂/EP receptor signalling for enhanced clathrin-mediated endocytosis. To determine if PGE₂ exerts its proviral effects during internalisation we supplemented MA104 cells with γ-linolenic acid (GLA), a precursor of arachidonic acid. Infection of supplemented cells with RV SA11 led to increased production of PGE₂ as monitored by ELISA. Confocal microscopy demonstrated that PGE₂ co-localises with the viroplasm-forming proteins, NSP5 and NSP2. Due to the known association of viroplasms as well as PGE₂ with lipid droplets, our results indicate a possible role for viroplasms in the production of RV-induced PGE₂. Replication kinetics showed that inhibitors, targeting the biosynthesis of PGE₂, had negative effects on RV yield, especially during the early stages of infection. Using flow cytometry and PGE₂ addback experiments, we show that PGE₂ enhances the attachment and internalisation of rotavirus in MA104 cells, indicating a possible role for PGE₂ during clathrin-mediated RV entry. Due to the well-known association between viroplasms and LDs, and the fact that LDs are production centres for the PGE₂, we next explored the possible role if any, of viroplasm components in the induction of PGE₂ production during RV infection. Transfection of HEK293 cells with plasmids, encoding the ORFs for NSP2 and NSP5 or both NSP2 and NSP5, showed that neither protein on their own, nor the formation of viroplasm-like structures was able to induce PGE₂ production. A MA104 cell line, stably expressing NSP5, was used to generate and characterize several SA11-based rescued rotaviruses (rSA11_aNSP5 and rSA11_aNSP5) with mutations in the α-helix within the C-terminal of NSP5. We demonstrate that a rSA11 with replaced hydrophobic amino acids in the C-terminal appeared to from less viroplasms compared to rSA11. This led to reduced replication of both rSA11_aNSP5 and rSA11_pNSP5, confirming the pivotal role of the α-helix within the C-terminal during RV replication. These mutations also affected the production of PGE₂ in HEK293 infected cells, although this is more likely due to decreased viral replication. Furthermore, we investigated how the introduced mutations affect NSP5 co-sedimentation with LD-associated protein, perilipin 2, and showed that the NSP5 mutations decreasing the hydrophobicity or abolishment of the α-helix changed the sedimentation profile. Our results indicate that individual viroplasm components or the formation of VLS do not induce PGE₂ in transfected cells, but that mutations in the C-terminal of NSP5 decrease PGE₂, most probably due to decreased replication. After showing that viroplasms mainly indirectly induce the production of PGE₂, we switched our focus to the role of Calcium (Ca²⁺) as it is essential for several cellular signalling and physiological processes, including the activation of cytoplasmic phospholipase A₂ (cPLA₂). This enzyme plays a role in lipid droplet (LD) biogenesis and is the rate-limiting enzyme in prostaglandin E₂ (PGE₂) biosynthesis. During rotavirus (RV) replication, NSP4 modulates the levels of cytoplasmic Ca²⁺ (cyto[Ca²⁺]) by a viroporin domain, which selectively conducts Ca²⁺ from endoplasmic reticulum (ER) stores to the cytoplasm. This modulation of cyto[Ca²⁺] is crucial for several viral process, including entry and assembly. We, therefore, investigated the role of the viroporin domain of NSP4 in the induction of PGE₂ production during RV infection. We show that RV infection of HEK293 cells increases the activity of cPLA₂, and that the chelation of cyto[Ca²⁺] decreases the activity of cPLA₂, leading to decreases in viral progeny and RNA yield. Mutations within the viroporin domain, which decreases the conductively of Ca²⁺, decreased the activity of cPLA₂ and subsequently affected PGE₂ levels as well as viral progeny and RNA yield. Our results indicate that the viroporin domain of NSP4 plays a role in the induction of PGE₂ production by increasing the activity of cPLA₂ in a Ca²⁺-dependent manner. Taken together, the data shows that PGE₂ is most likely induced in a NSP4-cyto[Ca²⁺]-cPLA₂-dependent manner enhancing RV internalisation. This enhanced internalisation increases viral yield, which could contribute indirectly to PGE₂ production by increasing the numbers of LDs and thus sites of PGE₂ production.
Description
Thesis (Ph.D.(Microbiology))--University of the Free State, 2022
Keywords
Rotavirus, internalisation, viroplasms, prostaglandin E2, cytoplasmic calcium, cytoplasmic phospholipase A2, NSP4, NSP5