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.
References
[1]
Lindenbach BD, Rice CM (2003) Molecular biology of flaviviruses. Adv Virus Res 59: 23–61. doi: 10.1016/s0065-3527(03)59002-9
Issur M, Geiss BJ, Bougie I, Picard-Jean F, Despins S, et al. (2009) The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure. RNA 15: 2340–2350. doi: 10.1261/rna.1609709
[4]
Tan BH, Fu J, Sugrue RJ, Yap EH, Chan YC, et al. (1996) Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. Virology 216: 317–325. doi: 10.1006/viro.1996.0067
[5]
Davidson AD (2009) Chapter 2. New insights into flavivirus nonstructural protein 5. Adv Virus Res 74: 41–101. doi: 10.1016/s0065-3527(09)74002-3
[6]
Bartenschlager R, Miller S (2008) Molecular aspects of Dengue virus replication. Future Microbiol 3: 155–165. doi: 10.2217/17460913.3.2.155
[7]
Murray CL, Jones CT, Rice CM (2008) Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis. Nat Rev Microbiol 6: 699–708. doi: 10.1038/nrmicro1928
[8]
Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, et al. (2009) Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5: 365–375. doi: 10.1016/j.chom.2009.03.007
[9]
Hanzal-Bayer MF, Hancock JF (2007) Lipid rafts and membrane traffic. FEBS Lett 581: 2098–2104. doi: 10.1016/j.febslet.2007.03.019
[10]
Jury EC, Flores-Borja F, Kabouridis PS (2007) Lipid rafts in T cell signalling and disease. Semin Cell Dev Biol 18: 608–615. doi: 10.1016/j.semcdb.2007.08.002
[11]
Aizaki H, Lee KJ, Sung VM, Ishiko H, Lai MM (2004) Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324: 450–461. doi: 10.1016/j.virol.2004.03.034
[12]
Mackenzie JM, Khromykh AA, Parton RG (2007) Cholesterol manipulation by West Nile virus perturbs the cellular immune response. Cell Host Microbe 2: 229–239. doi: 10.1016/j.chom.2007.09.003
[13]
Medigeshi GR, Hirsch AJ, Streblow DN, Nikolich-Zugich J, Nelson JA (2008) West Nile virus entry requires cholesterol-rich membrane microdomains and is independent of alphavbeta3 integrin. J Virol 82: 5212–5219. doi: 10.1128/jvi.00008-08
[14]
Chazal N, Gerlier D (2003) Virus entry, assembly, budding, and membrane rafts. Microbiol Mol Biol Rev 67: : 226–237, table of contents.
[15]
Manes S, del Real G, Martinez AC (2003) Pathogens: raft hijackers. Nat Rev Immunol 3: 557–568. doi: 10.1038/nri1129
[16]
Lee CJ, Lin HR, Liao CL, Lin YL (2008) Cholesterol effectively blocks entry of flavivirus. J Virol 82: 6470–6480. doi: 10.1128/jvi.00117-08
[17]
Reeves VL, Thomas CM, Smart EJ (2012) Lipid rafts, caveolae and GPI-linked proteins. Adv Exp Med Biol 729: 3–13. doi: 10.1007/978-1-4614-1222-9_1
[18]
Stuermer CA, Lang DM, Kirsch F, Wiechers M, Deininger SO, et al. (2001) Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble in noncaveolar plasma membrane microdomains defined by reggie-1 and -2. Mol Biol Cell 12: 3031–3045. doi: 10.1091/mbc.12.10.3031
[19]
Carter GC, Bernstone L, Sangani D, Bee JW, Harder T, et al. (2009) HIV entry in macrophages is dependent on intact lipid rafts. Virology 386: 192–202. doi: 10.1016/j.virol.2008.12.031
[20]
Simmons GE Jr, Taylor HE, Hildreth JE (2012) Caveolin-1 suppresses human immunodeficiency virus-1 replication by inhibiting acetylation of NF-kappaB. Virology 432: 110–119. doi: 10.1016/j.virol.2012.05.016
[21]
Huang JH, Lu L, Lu H, Chen X, Jiang S, et al. (2007) Identification of the HIV-1 gp41 core-binding motif in the scaffolding domain of caveolin-1. J Biol Chem 282: 6143–6152. doi: 10.1074/jbc.m607701200
[22]
Lin S, Wang XM, Nadeau PE, Mergia A (2010) HIV infection upregulates caveolin 1 expression to restrict virus production. J Virol 84: 9487–9496. doi: 10.1128/jvi.00763-10
[23]
Wang XM, Nadeau PE, Lin S, Abbott JR, Mergia A (2011) Caveolin 1 inhibits HIV replication by transcriptional repression mediated through NF-kappaB. J Virol 85: 5483–5493. doi: 10.1128/jvi.00254-11
[24]
Shi ST, Lee KJ, Aizaki H, Hwang SB, Lai MM (2003) Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77: 4160–4168. doi: 10.1128/jvi.77.7.4160-4168.2003
[25]
Mackenzie J (2005) Wrapping things up about virus RNA replication. Traffic 6: 967–977. doi: 10.1111/j.1600-0854.2005.00339.x
[26]
Rothwell C, Lebreton A, Young Ng C, Lim JY, Liu W, et al. (2009) Cholesterol biosynthesis modulation regulates dengue viral replication. Virology 389: 8–19. doi: 10.1016/j.virol.2009.03.025
[27]
Hailstones D, Sleer LS, Parton RG, Stanley KK (1998) Regulation of caveolin and caveolae by cholesterol in MDCK cells. J Lipid Res 39: 369–379.
[28]
Lorizate M, Krausslich HG (2011) Role of lipids in virus replication. Cold Spring Harb Perspect Biol 3: a004820. doi: 10.1101/cshperspect.a004820
[29]
Metzner C, Salmons B, Gunzburg WH, Dangerfield JA (2008) Rafts, anchors and viruses—a role for glycosylphosphatidylinositol anchored proteins in the modification of enveloped viruses and viral vectors. Virology 382: 125–131. doi: 10.1016/j.virol.2008.09.014
[30]
Puerta-Guardo H, Mosso C, Medina F, Liprandi F, Ludert JE, et al. (2010) Antibody-dependent enhancement of dengue virus infection in U937 cells requires cholesterol-rich membrane microdomains. J Gen Virol 91: 394–403. doi: 10.1099/vir.0.015420-0
[31]
Reyes-Del Valle J, Chavez-Salinas S, Medina F, Del Angel RM (2005) Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells. J Virol 79: 4557–4567. doi: 10.1128/jvi.79.8.4557-4567.2005
[32]
Vasquez Ochoa M, Garcia Cordero J, Gutierrez Castaneda B, Santos Argumedo L, Villegas Sepulveda N, et al. (2009) A clinical isolate of dengue virus and its proteins induce apoptosis in HMEC-1 cells: a possible implication in pathogenesis. Arch Virol 154: 919–928. doi: 10.1007/s00705-009-0396-7
[33]
Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, et al. (2009) Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol 83: 12895–12906. doi: 10.1128/jvi.00942-09
[34]
Gayen S, Chen AS, Huang Q, Kang C (2012) West Nile Virus (WNV) protease and membrane interactions revealed by NMR spectroscopy. Biochem Biophys Res Commun 423: 799–804. doi: 10.1016/j.bbrc.2012.06.043
[35]
Moreland NJ, Tay MY, Lim E, Rathore AP, Lim AP, et al. (2012) Monoclonal antibodies against dengue NS2B and NS3 proteins for the study of protein interactions in the flaviviral replication complex. J Virol Methods 179: 97–103. doi: 10.1016/j.jviromet.2011.10.006
[36]
Chen CJ, Kuo MD, Chien LJ, Hsu SL, Wang YM, et al. (1997) RNA-protein interactions: involvement of NS3, NS5, and 3′ noncoding regions of Japanese encephalitis virus genomic RNA. J Virol 71: 3466–3473.
[37]
Cui T, Sugrue RJ, Xu Q, Lee AK, Chan YC, et al. (1998) Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. Virology 246: 409–417. doi: 10.1006/viro.1998.9213
[38]
Radyukhin V, Fedorova N, Ksenofontov A, Serebryakova M, Baratova L (2008) Cold co-extraction of hemagglutinin and matrix M1 protein from influenza virus A by a combination of non-ionic detergents allows for visualization of the raft-like nature of the virus envelope. Arch Virol 153: 1977–1980. doi: 10.1007/s00705-008-0214-7
[39]
Heaton NS, Perera R, Berger KL, Khadka S, Lacount DJ, et al. (2010) Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis. Proc Natl Acad Sci U S A 107: 17345–17350. doi: 10.1073/pnas.1010811107
[40]
Brown DA (2006) Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda) 21: 430–439. doi: 10.1152/physiol.00032.2006
[41]
Ravid D, Leser GP, Lamb RA (2010) A role for caveolin 1 in assembly and budding of the paramyxovirus parainfluenza virus 5. J Virol 84: 9749–9759. doi: 10.1128/jvi.01079-10
[42]
Bustos-Arriaga J, Garcia-Machorro J, Leon-Juarez M, Garcia-Cordero J, Santos-Argumedo L, et al. (2011) Activation of the innate immune response against DENV in normal non-transformed human fibroblasts. PLoS Negl Trop Dis 5: e1420. doi: 10.1371/journal.pntd.0001420
[43]
Morens DM, Halstead SB, Repik PM, Putvatana R, Raybourne N (1985) Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microbiol 22: 250–254.
[44]
Garcia-Cordero J, Ramirez HR, Vazquez-Ochoa M, Gutierrez-Castaneda B, Santos-Argumedo L, et al. (2005) Production and characterization of a monoclonal antibody specific for NS3 protease and the ATPase region of Dengue-2 virus. Hybridoma (Larchmt) 24: 160–164. doi: 10.1089/hyb.2005.24.160
[45]
Garcia-Cordero J, Carrillo-Halfon S, Leon-Juarez M, Romero-Ramirez H, Valenzuela-Leon P, et al. (2014) Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2. Immunol Invest 43: 28–40. doi: 10.3109/08820139.2013.833622
