Modulation of Zika virus replication via glucosylceramide 1 synthase and glycosphingolipids

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Submission ID :
ESPR387
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Abstract: :

Background:  The enveloped positive-sense RNA viruses such as Zika virus (ZIKV) need host cell membrane lipids to successfully replicate. The nature of the lipids and the replication step(s) where the lipids are utilized often vary amongst viruses. We previously reported that hepatitis C virus (HCV) recruits host glycosphingolipids to the viral replication complex to facilitate HCV genome replication.

Objective: To understand the utilization of glycosphingolipids in ZIKV and other RNA virus replication.

Design/Methods:  Multiple methods were employed, including ZIKV plaque assays, quantitative real-time PCR, immunoblotting of ZIKV and host proteins, indirect immunofluorescence, sucrose cushion purification of ZIKV particles, negative staining and electron microscopy of ZIKV particles, and determination of sphingolipid levels measured by high-performance liquid chromatography/tandem mass spectrometry.

Results:  Lipidomic analysis of partially purified ZIKV particle envelope shows significant enrichment in distinct sphingolipid species, implying that sphingolipids are incorporated into ZIKV particle to facilitate viral replication. To determine the role of sphingolipids in ZIKV replication, we leveraged a panel of sphingolipid-deficient cell lines. Notably, knockout of a crucial glycosphingolipid metabolic gene (GCSKO), or pharmacological inhibition of GCS enzyme, resulted in a marked decrease in ZIKV titer (Figure 1). Interestingly, glycosphingolipid deficiency has a minimal impact on ZIKV genome replication, implying that these lipids are required in post ZIKV genome replication steps. Notably, our data indicate that glycosphingolipid deficiency negatively impacts ZIKV particle assembly. In addition, we show that ZIKV core protein colocalizes with lipid droplets, and pharmacological inhibition of an enzyme involved in lipid droplet production leads to a significant decrease in ZIKV replication (Figure 2). These findings imply that early steps in ZIKV assembly occur on lipid droplets. Finally, glycosphingolipid deficiency also leads to a marked decrease in Dengue virus (DENV) titer.

Conclusions:  Altogether, our study implies that ZIKV and DENV harness the function of glycosphingolipids, and/or their proteins, to facilitate infectious virus particle production.

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Albany Medical College
Albany Medical Center, Albany NY
Albany Medical College
Albany College of Pharmacy and Health Sciences
National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
Albany Medical College
National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan
Albany Medical College

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