Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments

Abstract

BackgroundThe replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells.Methodology/Principal FindingsEntry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl β-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island.ConclusionsTogether our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.

Potential entry inhibitors of the envelope protein (E2) of Chikungunya virus: in silico structural modeling, docking and molecular dynamic studies

“…4 The ribbon image showing superimposition of the modeled E2 protein (purple color) and the crystallized immature E1-E2-E3complex of Chikungunya virus [46] (PDB Id: 3n40) (green color) (color figure online) by Hunt and colleagues reported that Bafilomycin A1 was found to block the expression of a virus-encoded reporter gene of sindbis virus in both infection and transfection of the BHK cells [18]. Further, another study reported limited inhibitory effect of bafilomycin in post CHIKV infection in HEK239T cells [5]. The inhibitory effect of bafilomycin has also been reported previously for other viruses like influenza [17].…”

Chikungunya and Its Interaction With the Host Cell

“…A detailed entry mechanism description is not available so far. Some authors implicate a clathrin-dependent pathway [64,66], whereas other authors have proposed that CHIKV enters by a clathrin-independent, but Eps15-dependent pathway [65]. These later authors also have found evidence that multiple pathways maybe employed by CHIKV to enter into cells, as already proposed with dengue virus (DENV) [67,68].…”

“…Viral replication is initiated by attachment of viral envelope to host cell receptors [63], followed by low pH-mediated membrane fusion and delivery of the viral nucleocapsid into the cytoplasm (Fig. 1) [64,65]. A detailed entry mechanism description is not available so far.…”

Recent progress on chikungunya virus research

“…Although specific host receptors for CHIKV are still to be determined, CHIKV is believed to enter cells by receptor-mediated pathway through E2 glycoprotein (Weber et al, 2017) (Figure 4B), and it is internalized either via clathrin-mediated endocytosis or a clathrin-independent Eps-15-dependent endocytosis pathway (Hoornweg et al, 2016), like other alphaviruses. Eps-15 is a member of clathrin-coated pits (Bernard et al, 2010). With the progress of infection, the endosome become acidic, inducing the dissociation of E2-E1 heterodimer and a conformation change of E1 membrane fusion protein, allowing host cell membrane and virus envelop to fuse, followed by the release of viral nucleocapsids into the cytoplasm (Gibbons et al, 2004;Bernard et al, 2010).…”

“…Eps-15 is a member of clathrin-coated pits (Bernard et al, 2010). With the progress of infection, the endosome become acidic, inducing the dissociation of E2-E1 heterodimer and a conformation change of E1 membrane fusion protein, allowing host cell membrane and virus envelop to fuse, followed by the release of viral nucleocapsids into the cytoplasm (Gibbons et al, 2004;Bernard et al, 2010). The nucleocapsid protein will then bind to the ribosome (considered to be cellular uncoating factor) to initiate uncoating, resulting in the release of genomic RNA into the cytoplasm (Singh and Helenius, 1992).…”