Abstract
VP22 is a structural protein of the herpes simplex virus and has been reported to possess unusual trafficking properties. Here we examined the mechanism of cellular uptake of VP22 using a fusion protein between the C-terminal half of VP22 and green fluorescent protein (GFP). Adsorption of VP22-GFP onto a cell surface required heparan sulfate proteoglycans and basic amino acids, in particular, Arg-164 of VP22. Inhibitor treatment, RNA interference, expression of dominant-negative mutant genes, and confocal microscopy all indicated that VP22-GFP enters cells through an endocytic pathway independent of clathrin and caveolae but dependent on dynamin and Arf6 activity. As with CD59 (a lipid raft marker), cell-surface VP22-GFP signals were resistant to Triton X-100 treatment but only partially overlapped cell-surface CD59 signals. Furthermore, unlike other lipid raft-mediated endocytic pathways, no Rho family GTPase was required for VP22-GFP internalization. Internalized VP22 initially entered early endosomes and then moved to lysosomes and possibly recycling endosomes.
Highlights
Showed that these short peptide-type PTDs and the proteins associated with them are most likely to be incorporated into cells via some endocytic mechanism [7, 8]
To determine which basic amino acids of VP22.C1 are required for interactions between heparan sulfate and VP22-green fluorescent protein (GFP), the basic amino acids of VP22.C1 were systematically replaced with alanine or glutamic acid, and we examined spectrofluorometrically whether mutant VP22-GFP proteins were effectively incorporated into cells (Fig. 2F)
We have shown that cell-surface binding and cellular internalization of VP22 require interactions between cell-surface heparan sulfate and basic amino acids in the N-terminal region of VP22.C1 (C-terminal half of VP22), which includes Arg-164, and that VP22 is internalized through a lipid raft-mediated endocytic pathway independent of clathrin, caveolae, and Rho family GTPases but dependent on dynamin and Arf6
Summary
Drugs and Antibodies—Alexa Fluor 647-labeled transferrin, phalloidin-labeled rhodamine, and Hoechst 33342 were purchased from Molecular Probes. After being fixed with 2– 4% paraformaldehyde in PBS for 15 min at room temperature unless indicated otherwise, the cells were washed twice with ice-cold PBS and mounted in VECTASHIELD mounting medium (Vector Laboratories) This was followed by cell visualization with an Olympus Fluoview FV1000 confocal microscopy system. After addition of the indicated concentrations of VP22-GFP or 2 g/ml anti-heparan sulfate antibody in serum-free DMEM, cells were further incubated on ice for 1 h, washed twice with ice-cold PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and visualized by confocal microscopy. Immunofluorescence—Cells were cultured in 12-well culture plates on a glass coverslip, incubated with protein at the specified concentrations in serum-free DMEM for the indicated times, washed twice with ice-cold PBS, and fixed with 4% paraformaldehyde at room temperature for 15 min. The following primers were used: Arf6, 5Ј-ATCAATGACCGGGAGATGAG-3Ј (forward) and 5Ј-AGGGCTGCACATACCAGTTC-3Ј (reverse); Cdc42, 5Ј-CGATGGTGCTGTTGGTAAAA-3Ј (forward) and 5Ј-TCCTCTTGCCCTGCAGTATC-3Ј (reverse); RhoA, 5Ј-CGGTCTGGTCTTCAGCTACC-3Ј (forward) and 5Ј-CCATCACCAACAATCACCAG-3Ј reverse; and -actin, 5Ј-CACACTGTGCCCATCTACGA-3Ј (forward) and 5Ј-GCCATCTCTTGCTCGAAGTC-3Ј (reverse)
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