title: 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.
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y Estudios Avanzados del IPN, México City, México; Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas IPN, México City, México.
authors.forename: Julio
authors.initials: J
authors.lastname: García Cordero
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y Estudios Avanzados del IPN, México City, México.
authors.forename: Moisés
authors.initials: M
authors.lastname: León Juárez
authors.affiliation: Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas IPN, México City, México.
authors.forename: Jorge A
authors.initials: JA
authors.lastname: González-Y-Merchand
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y Estudios Avanzados del IPN, México City, México.
authors.forename: Leticia
authors.initials: L
authors.lastname: Cedillo Barrón
authors.affiliation: Laboratorio de Inmunología, Facultad de Estudios Superiores Iztacala Universidad Autónoma de México, Tlalnepantla Estado de México, México.
authors.forename: Benito
authors.initials: B
authors.lastname: Gutiérrez Castañeda
mesh_terms: D051242:Caveolin 1 / Q000378:metabolism*; D002460:Cell Line; D003716:Dengue Virus / Q000502:physiology*; D003902:Detergents / Q000737:chemistry; D042783:Endothelial Cells / Q000378:metabolism* / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism* / Q000821:virology; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D053586:Virus Internalization; D014779:Virus Replication; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D051242:Caveolin 1; D003902:Detergents; D008565:Membrane Proteins; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C495540:CAV1 protein, human; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C106354:flotillins