Inhibition of Cytosolic Phospholipase A₂α Impairs an Early Step of Coronavirus Replication in Cell Culture

Abstract

Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase Aα (cPLAα) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLAα activity, which produces lysophospholipids (LPLs) by cleaving at the -2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i)viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLAα inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the and families, while members of the were not affected. Taken together, the study provides evidence that cPLAα activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development. Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2α, which releases fatty acid from the -2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments in which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication, and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLAα inhibitor.

COVID-19 infection results in alterations of the kynurenine pathway and fatty acid metabolism that correlate with IL-6 levels and renal status

“…Small molecule metabolites are essential for viral infection, because they provide building blocks that a rapidly proliferating virus requires to assemble its nucleic acids, proteins (including capsid proteins that mediate infection of new cell targets), and membrane. Indeed, viral infections mobilize free fatty acids to support viral capsid-associated membrane formation, which was described for other coronaviruses and is explained, in part, by activation of phospholipase A2, a target amenable to pharmacological intervention (24)(25)(26). The current study provides the first comprehensive targeted and untargeted metabolomics analysis of sera from COVID-19 patients, stratified by circulating levels of IL-6, and correlated to inflammatory markers and renal function.…”

“…Indeed, a key finding in the current study was elevated long-chain free fatty acids in COVID-19 subjects. Phospholipase A2 (PLA2), which cleaves the fatty acid in the sn-2 position of phospholipids, is required for replication of other related viruses such as MERS-CoV and HCoV-229E (25). Elevated levels of long-chain polyunsaturated fatty acids (PUFA), which are highly enriched in the sn-2 position of phospholipids, suggests that this pathway may be upregulated during SARS-CoV-2 infection.…”

“…Increased PLA2 activity is associated with increased production of bioactive lipids by metabolism of omega-6 PUFAs and lysophosphatidylcholine. Some of the resulting metabolites (e.g., eicosanoids, oxylipins) may help propagate infection and thrombo-inflammatory complications (59), by directly supporting synthesis of viral capsid membranes (24)(25)(26), by targeting All rights reserved. No reuse allowed without permission.…”

Small-molecule inhibitors as potential therapeutics and as tools to understand the role of phospholipases A2

“…Another study has provided evidence that GIVA cPLA 2 activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development [35]. The inhibition of GIVA cPLA 2 activity by the low-molecular-weight inhibitor pyrrolidine-2 (RSC-3388) has profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells.…”

Comparison of broad-spectrum antiviral activities of the synthetic rocaglate CR-31-B (−) and the eIF4A-inhibitor Silvestrol

“…After 1 h, the inoculum was removed and cells were incubated with fresh medium containing the inhibitor at increasing concentrations. Supernatants were collected at 24 h post infection (hpi; HCoV-229E, MERS-CoV) or 3 dpi (LASV, CCHF) and virus titers were analyzed by virus plaque assay (Müller et al, 2018b) or immunofocus assay as described before (Günther et al, 2004). To calculate EC 50 values, the virus titer determined for virusinfected cells treated with DMSO only was set to 100% and titers obtained for treated cells were normalized to this value.…”