Phospholipase A2 activity during the replication cycle of the flavivirus West Nile virus.

Susann Liebscher,Andrea Mikulasova,Jason M Mackenzie,Rebecca L Ambrose,Leah K Gillespie,Malcolm J Mcconville,Turgut E Aktepe,Dedreia Tull,Julia E Prier,Thusitha W T Rupasinghe,Adam J Lopez-Denman,Richard J Kuhn

doi:10.1371/journal.ppat.1007029

Susann Liebscher, Andrea Mikulasova + Show 10 more

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https://doi.org/10.1371/journal.ppat.1007029

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Abstract

Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.

Highlights

Cellular lipids play a vital role in the replication of flaviviruses; forming the membranous microenvironments surrounding the replication complex (RC), structural components of the virus particle, and providing a source of metabolic precursors for ATP synthesis in the host cell [1,2,3,4,5,6,7]
Positive-sense RNA viruses remodel the host cytoplasmic membrane architecture to induce the formation of membranous organelles termed viral replication complexes
Our results indicate that the replication of West Nile virus (WNV) strain Kunjin virus (WNVKUN) is dependent on and appears to up-regulate the activity of phospholipase A2 (PLA2) to produce lyso-PChol from PChol, observations that are consistent with a recent lipidomic study of dengue-infected mosquito cells [6]

Summary

Introduction

Cellular lipids play a vital role in the replication of flaviviruses; forming the membranous microenvironments surrounding the replication complex (RC), structural components of the virus particle, and providing a source of metabolic precursors for ATP synthesis in the host cell [1,2,3,4,5,6,7]. We have previously shown a strict requirement for cholesterol and ceramide during WNV strain Kunjin virus (WNVKUN) replication [3, 10], the utilisation of ceramide was different to that we observed for DENV Extending these studies further other groups have performed lipidomic analyses on DENV-infected mosquito cells and whole WNV virions, and identified discrete changes and requirements of specific lipid groups during infection [5, 6]. It is evident from multiple previous studies that the intimate interactions between flaviviruses and membrane platforms within the endoplasmic reticulum (ER) are the governing connections that establish and facilitate efficient virus replication. There is a need to interrogate the critical requirements and interactions that occur on these membranes platforms as the development of interventions that can diminish this interface may severely restrict and hamper efficient virus replication

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