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

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.

The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis

“…Enrichment in SLs and a reduction of PC levels are characteristics of liquid ordered membrane microdomains or membrane rafts (7,9,18,56). Since membrane raft-like microdomains, including those located in the ER (57), are usually cholesterol enriched (7,9,18,56), our findings could be connected to the role(s) of membrane microdomains enriched in cholesterol in flavivirus infections (28,31,(58)(59)(60). SL-enriched membrane microdomains have functional properties directly linked to their lipid composition, such as coalescence and the promotion of budding processes during virus assembly (7,9) or exosome biogenesis (18).…”

Dengue Virus Serotype 2 and Its Non-Structural Proteins 2A and 2B Activate NLRP3 Inflammasome

“…The knockout was performed according to the protocol (37). lentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid # 52961; http://n2t.net/addgene:52961; RRID:Addgene_52961 • C for 45 s and more tubes were placed on ice for 2 min.…”

Recombinant Dengue virus protein NS2B alters membrane permeability in different membrane models

“…These characteristics include the hydrophilic segment, which is required for the cofactor activity of viral protease NS3, and the 3 hydrophobic regions that are thought to be responsible for membrane association and to generate the MDA. In addition, we previously demonstrated that NS2B is localized in cellular membranes (particularly in lipid rafts) due to the hydrophobic regions [ 20 ]. Data from a subsequent study suggested that the NS2B protein contains alpha-helical transmembrane domains that direct folds within micelles, indicating the ability of this protein to associate with membranes [ 21 ].…”