Cellular Internalization of Green Fluorescent Protein Fused with Herpes Simplex Virus Protein VP22 via a Lipid Raft-mediated Endocytic Pathway Independent of Caveolae and Rho Family GTPases but Dependent on Dynamin and Arf6

doi:10.1074/jbc.M703810200

Cellular Internalization of Green Fluorescent Protein Fused with Herpes Simplex Virus Protein VP22 via a Lipid Raft-mediated Endocytic Pathway Independent of Caveolae and Rho Family GTPases but Dependent on Dynamin and Arf6^*

Kenji Nishi¹ and
Kaoru Saigo²

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

1 To whom correspondence may be addressed. Tel.: 81-3-5841-3044; Fax: 81-3-5841-3044; E-mail: kennishi{at}biochem.s.u-tokyo.ac.jp.
2 To whom correspondence may be addressed. Tel.: 81-3-5841-3044; Fax: 81-3-5841-3044; E-mail: saigo{at}biochem.s.u-tokyo.ac.jp.

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.

Footnotes

↵³ The abbreviations used are: PTDs, protein transduction domains; GFP, green fluorescent protein; MβCD, methyl-β-cyclodextrin; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; RNAi, RNA interference; siRNAs, small interfering RNAs; GAG, glycosaminoglycan.
↵⁴ K. Nishi and K. Saigo, unpublished data.
↵⁵ The sequences of the primers used for mutagenesis are available upon request.
↵⁶ K. Ui-Tei, K. Nishi, and K. Saigo, unpublished data.
↵^* This work was supported in part by a special coordination fund for promoting science and technology and by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3.
- Received May 9, 2007.
- Revision received July 2, 2007.