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

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.

Shaping the flavivirus replication complex: It is curvaceous!

“…We speculate that the viral protein NS4A (based on its predicted topology, structure, and membrane remodelling capacity [Roosendaal et al, , Miller et al, ]) induces membrane curvature while interacting with the host RTN3.1A protein (Aktepe et al, ). We suggest that the biogenesis and recruitment of the cone‐shaped lipids ceramide (Aktepe et al, ) and lyso‐PChol (Liebscher et al, ) stabilises and provides the required curvature for the formation of the VP. Viral and host proteins supporting the VP neck structure (an integral component of the VP that allows for the passage of nucleotides, proteins, and viral RNA) is currently unknown; however, components of the ESCRT complex may be recruited to aid in the stabilisation of the neck (Barajas et al, , Barajas et al, , Diaz et al, )…”

“…Both these studies observed a significant decrease in phosphatidylcholine (PChol) abundance with an increase in lyso-PChol. This change is indicative of increased activity of the host enzyme Phospholipase A2, a role that has been recently shown for at least WNV (Liebscher et al, 2018). In contrast to ceramide, lyso-PChol exhibits the capacity to induce positive membrane curvature due to its relatively large hydrophilic head group in relation to its hydrocarbon tail (Fuller & Rand, 2001).…”

“…We speculate that the viral protein NS4A (based on its predicted topology, structure, and membrane remodelling capacity [Roosendaal et al, 2006, Miller et al, 2007) induces membrane curvature while interacting with the host RTN3.1A protein (Aktepe et al, 2017). We suggest that the biogenesis and recruitment of the cone-shaped lipids ceramide (Aktepe et al, 2015) and lyso-PChol (Liebscher et al, 2018) (Yi et al, 2012). This characterisation of DNAJC14 highlights the importance of host factors in regulating flavivirus replication and the protein-protein and protein-lipid interactions that drive and shape RC formation and stabilisation.…”

“…Two lipidomic analyses on WNV‐infected mammalian cells and DENV‐infected mosquito cells have revealed a distinct modulation of the phospholipid landscape upon infection (Liebscher et al, ; Perera et al, ). Both these studies observed a significant decrease in phosphatidylcholine (PChol) abundance with an increase in lyso‐PChol.…”

Targeting host lipid flows: Exploring new antiviral and antibiotic strategies

“…For instance, HCV infection has been discovered to increase cellular levels of cholesterol and sphingolipids (SLs) and to modify the phospholipid metabolism leading to an accumulation of phosphatidylcholine (PC) and triglyceride with longer and polyunsaturated fatty acid (FA) chains (Diamond et al, 2010;Gullberg et al, 2018;Hofmann et al, 2018;Roe, Kensicki, Mohney, & Hall, 2011). Lipidomics analysis of cells infected with other related flaviviruses such as DENV and WNV has revealed a marked increase in FAs, glycerophospholipids (GPLs), and SL content (Chotiwan et al, 2018;Perera et al, 2012) and has allowed to identify specific markers of infection, for example, lysophosphatidylcholine (Liebscher et al, 2018).…”