Caveolin-1 in Lipid Rafts Interacts with Dengue Virus NS3 during Polyprotein Processing and Replication in HMEC-1 Cells

Julio García Cordero,Leticia Cedillo Barrón,Jorge A González-Y-Merchand,Moisés León Juárez,Benito Gutiérrez Castañeda,Tianyi Wang

doi:10.1371/journal.pone.0090704

Julio García Cordero, Leticia Cedillo Barrón + Show 4 more

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https://doi.org/10.1371/journal.pone.0090704

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Abstract

Lipid rafts are ordered microdomains within cellular membranes that are rich in cholesterol and sphingolipids. Caveolin (Cav-1) and flotillin (Flt-1) are markers of lipid rafts, which serve as an organizing center for biological phenomena and cellular signaling. Lipid rafts involvement in dengue virus (DENV) processing, replication, and assembly remains poorly characterized. Here, we investigated the role of lipid rafts after DENV endocytosis in human microvascular endothelial cells (HMEC-1). The non-structural viral proteins NS3 and NS2B, but not NS5, were associated with detergent-resistant membranes. In sucrose gradients, both NS3 and NS2B proteins appeared in Cav-1 and Flt-1 rich fractions. Additionally, double immunofluorescence staining of DENV-infected HMEC-1 cells showed that NS3 and NS2B, but not NS5, colocalized with Cav-1 and Flt-1. Furthermore, in HMEC-1cells transfected with NS3 protease, shown a strong overlap between NS3 and Cav-1, similar to that in DENV-infected cells. In contrast, double-stranded viral RNA (dsRNA) overlapped weakly with Cav-1 and Flt-1. Given these results, we investigated whether Cav-1 directly interacted with NS3. Cav-1 and NS3 co-immunoprecipitated, indicating that they resided within the same complex. Furthermore, when cellular cholesterol was depleted by methyl-beta cyclodextrin treatment after DENV entrance, lipid rafts were disrupted, NS3 protein level was reduced, besides Cav-1 and NS3 were displaced to fractions 9 and 10 in sucrose gradient analysis, and we observed a dramatically reduction of DENV particles release. These data demonstrate the essential role of caveolar cholesterol-rich lipid raft microdomains in DENV polyprotein processing and replication during the late stages of the DENV life cycle.

Highlights

Dengue viruses (DENVs) are enveloped, positive-sense RNA viruses that belong to the Flaviviridae family.dengue virus (DENV) initiate their life cycle through receptor-mediated endocytosis at the cellular membrane
When cellular cholesterol was depleted by methyl-beta cyclodextrin treatment after DENV entrance, lipid rafts were disrupted, NS3 protein level was reduced, besides Cav-1 and NS3 were displaced to fractions 9 and 10 in sucrose gradient analysis, and we observed a dramatically reduction of DENV particles release
To confirm the cellular distribution of the lipid raft protein markers Cav-1 and flotillin 1 (Flt-1) in our model, HMEC-1cells were stained with anti-Cav-1 and anti-Flt-1 antibodies

Summary

Introduction

Dengue viruses (DENVs) are enveloped, positive-sense RNA viruses that belong to the Flaviviridae family.DENVs initiate their life cycle through receptor-mediated endocytosis at the cellular membrane. The conformation of the viral envelope protein changes to promote the release of the genome into the cytoplasm. The genome is translated into a large polyprotein that is proteolytically processed to yield three structural proteins (envelope protein, membrane precursor protein, and capsid) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [1,2]. NS5 contains a methyltransferase domain and an RNA polymerase domain [3,4,5].Dengue virus induces the remodeling and redistribution of distinct membrane structures to obtain a platform for viral RNA replication, assembly, and spreading, [6,7]. Image analyses have shown physical linkages between the sites of DENV replication and assembly [8]

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