Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrin-mediated endocytosis

Abstract

Entry of dengue virus 2 (DENV-2) into Aedes albopictus mosquito C6/36 cells was analysed using biochemical and molecular inhibitors, together with confocal and electron microscopy observations. Treatment with monodansylcadaverine, chlorpromazine, sucrose and ammonium chloride inhibited DENV-2 virus yield and protein expression, whereas nystatin, a blocker of caveolae-mediated endocytosis, did not have any effect. Using confocal microscopy, co-localization of DENV-2 E glycoprotein and the marker protein transferrin was observed at the periphery of the cytoplasm. To support the requirement of clathrin function for DENV-2 entry, overexpression of a dominant-negative mutant of Eps15 in C6/36 cells was shown to impair virus entry. The disruption of actin microfilaments by cytochalasin D also significantly affected DENV-2 replication. In contrast, microtubule disruption by colchicine treatment did not impair DENV-2 infectivity, suggesting that DENV-2 does not require transport from early to late endosomes for successful infection of mosquito cells. Furthermore, using transmission electron microscopy, DENV-2 particles of approximately 44-52 nm were found attached within electron-dense invaginations of the plasma membrane and in coated vesicles that resembled those of clathrin-coated pits and vesicles, respectively. Together, these results demonstrate for the first time that DENV-2 enters insect cells by receptor-mediated, clathrin-dependent endocytosis, requiring traffic through an acidic pH compartment for subsequent uncoating and completion of a productive infection. INTRODUCTIONDengue virus (DENV) is an arthropod-borne member of the genus Flavivirus, family Flaviviridae, and can cause either dengue fever, a relatively benign, self-limited, febrile disease, or the severe and potentially fatal disease dengue haemorrhagic fever/dengue shock syndrome. The virion is an enveloped particle containing a single, positive-stranded RNA and three structural proteins: the capsid protein (C), a small membrane protein (M) and the envelope glycoprotein (E). There are four serotypes of DENV (DENV-1 to -4), which circulate in nature between their vectors, the mosquitoes Aedes aegypti and Aedes albopictus, and the vertebrate hosts. Currently, it is estimated that the virus is endemic in more than 100 countries, producing about 50 million infections each year (Gubler, 2002). Despite the importance and increasing incidence of DENV as a human pathogen, there is no antiviral chemotherapy and little is known about its cell biology.The initial event in virus infection involves virion attachment to cell-surface-specific receptors followed by internalization into the cytoplasm. After binding to the host cell, enveloped viruses exploit two main pathways for entry and uncoating: direct fusion between the viral envelope and the plasma membrane, and endocytosis. The most common and well-known mode of endocytosis is the classical clathrin-mediated pathway with penetration in early or late endosomes where the exposure to low pH conditions triggers ...

C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes

“…MDC is a pharmacological inhibitor of receptor-mediated endocytosis [18,21], while filipin, which binds cholesterol in the plasma membrane, impairs the invagination and subsequent internalisation of caveolae [23,24]. Under our experimental conditions, pretreatment of HAEC with MDC (43 mmol/l) for 30 min completely blocked the uptake of Alexa Fluor 488-labelled C-peptide (p<0.01 vs control) (Fig.…”

“…Isolation of endosomes and analysis of C-peptide content To identify early endosomes, we followed the internalisation of Alexa Fluor 488-conjugated human transferrin, a ligand known to enter the cells by clathrin-mediated endocytosis and a specific marker of early endosomes [21], in the presence of Alexa Fluor 546-labelled C-peptide in UASMC in the incubator for 10 min. Endosomes were isolated from other subcellular compartments by centrifugation (modified from [22]) and an aliquot of both the pellet and the endosome-containing supernatant fraction was used to quantitate Alexa Fluor 488-transferrin and Alexa Fluor 546-C-peptide fluorescence spectra by using a scanning spectrofluorometer.…”

Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library

“…During a blood meal of a dengue-infected person by Aedes mosquito, dengue virus (DENV) is ingested into the mosquito's midgut. It is generally believed that the mosquito midgut carries important cellular membrane receptors that facilitate viral entry through receptor-mediated endocytosis [2,3], enabling replication and exocytosis, followed by DENV disseminations to salivary glands for transmission to humans [4,5].…”

Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells

“…In past decades, the entry of JEV into host cells, either mammalian or mosquito, was demonstrated to be significantly influenced by treatment with bafilomycin A1 (Andoh et al, 1998;Nawa, 1998). In fact, a number of flaviviruses like West Nile virus (Jiang et al, 2005), DENV (Acosta et al, 2008) and JEV (Chai et al, 2013), have been known to infect host cells via this pathway. Furthermore, the cationic amphiphilic drug chlorpromazine can interfere with the entry process (Chai et al, 2013;Nawa et al, 2003), revealing that the mode of infection is receptormediated endocytosis and is clathrin-dependent.…”

“…Orthobunyavirus, one of the encephalitic arboviruses, reportedly infects mammalian host cells via clathrin-mediated endocytosis as its primary mechanism (Hollidge et al, 2012). This mechanism for infecting host cells is also implemented by various mosquito-borne flaviviruses (Acosta et al, 2008;Chu & Ng, 2004). Among them the Japanese encephalitis virus (JEV), which infects host (either mammalian or mosquito) cells via receptor-mediated endocytosis (Andoh et al, 1998;Nawa, 1998), has also been confirmed to be clathrindependent (Chai et al, 2013;Yang et al, 2013), except for certain unusual cases (Kalia et al, 2013).…”