Modification of the Host Cell Lipid Metabolism Induced by Hypolipidemic Drugs Targeting the Acetyl Coenzyme A Carboxylase Impairs West Nile Virus Replication

Abstract

West Nile virus (WNV) is a mosquito-borne neurotropic flavivirus responsible for recurrent outbreaks of meningitis and encephalitis affecting humans, horses, and birds in Africa, Europe, Asia, Oceania, and America (1). A great effort has been devoted in the past several years to decipher the molecular biology of WNV and its interaction with the host immune system (2, 3). Nevertheless, no licensed vaccine or therapy for human use against this pathogen is yet available.The flavivirus life cycle (including that of WNV) is intimately associated with host cell lipids. The replication of the viral genomic RNA and flavivirus nascent virion assembly take place in modified membranes from the endoplasmic reticulum (4-7). To build an adequate microenvironment to support viral replication and particle biogenesis, flaviviruses rearrange host cell lipid metabolism by promoting the synthesis and accumulation of specific cellular lipids (i.e., fatty acids, glycerophospholipids [GPLs], sphingolipids [SLs], and cholesterol) (8-15). This makes the pharmacological manipulation of cellular lipids an attractive antiviral strategy against WNV and related flaviviruses (13,14,16,17).The first steps of lipid biogenesis involve the synthesis and elongation of fatty acids, which provide the building blocks for the synthesis of more-complex lipids. Hence, fatty acid synthesis and elongation have become key targets for antiviral therapy (13,18,19). Regarding the flaviviruses, the pharmacological blockage of the fatty acid synthase FASN (which catalyzes the synthesis of palmitate from acetyl coenzyme A [acetyl-CoA] and malonylCoA into long-chain saturated fatty acids) reduced the viral replication (11, 13). The enzyme preceding FASN in the fatty acid biosynthetic route is the acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Due to its rate-limiting role in fatty acid synthesis, ACC is currently a target of increasing interest within the pharmacological industry (20, 21). However, to our knowledge, the involvement of ACC in the replication of WNV, or other related flaviviruses, has not yet been evaluated.In this work, we have shown that ACC inhibitors alter the cellular lipid composition and reduce the levels of WNV infection in cultured cells. Furthermore, infection by Usutu virus (USUV; a related emerging flavivirus [22]) was also inhibited by the drugs used. Our results point to ACC as a potential druggable antiviral target against WNV and related flaviviruses. MATERIALS AND METHODSCells, viruses, infections, and virus titrations. All infectious virus manipulations were performed in biosafety level 3 (BSL-3) facilities. Vero, HeLa, and Neuro2a (N2a) cell lines were cultured as described previously (10, 23). Cells were incubated with the corresponding virus, WNV strain NY99 or USUV strain SAAR-1776 (24), for 1 h at 37°C; viral inoculum was removed; and infected cells were incubated in culture medium containing 1% fetal bovine serum (time that was considered 1 h postinfection [p.i.]).

Host sphingomyelin increases West Nile virus infection in vivo

“…Considering that SM could play a structural role in WNV particles ( 8 ), the expected reduction on SM content due to SMS inhibition could result in a reduced availability of SM for viral envelopment, thus causing a decrease in the infectious viral particles produced. Similar effects have been observed in other viral models using other drugs that also block lipid synthesis (48)(49)(50). The amount of cellassociated viral RNA was also measured in cells treated with the inhibitors ( Fig.…”

Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond

“…However, FASN inhibitors can cause severe anorexia and weight loss 212 , and inhibition of ACC appears to have a higher clinical potential for the treatment of viral infections 212 . The ACC inhibitors TOFA (5-(tetradecyloxy)-2-furoic acid) and MEDICA 16 (3,3,14,14-tetramethylhexadecanedioic acid) induced a dose-dependent reduction of WNV and Usutu virus replication 213 .…”

Antiviral Activity of Nordihydroguaiaretic Acid and Its Derivative Tetra- O -Methyl Nordihydroguaiaretic Acid against West Nile Virus and Zika Virus

“…Immunofluorescence and confocal microscopy were performed as described previously (17,44). Nuclei were stained with To-Pro-3 (Life Technologies), and images were acquired using a Leica TCS SPE confocal laser scanning microscope and an HCX PL Apo 63ϫ/1.4 oil immersion objective.…”

“…For quantification of cell-associated viral RNA, supernatants from infected cells were removed and total RNA was extracted from cell monolayers using TRIzol reagent (Life Technologies). The amount of viral RNA was determined by real-time fluorogenic reverse transcriptase PCR (RT-PCR) according to previously published protocols (44,45). As a control, the amount of rRNA 18S in RNA samples from cell monolayers was determined as described previously (46).…”