J Virol 78(22): 12259-12267

Uptake Pathway of Polyomavirus via Ganglioside GD1a

Department of Pathology, Harvard Medical School, Boston, Massachusetts

^{Corresponding author. Mailing address: Department of Pathology, Harvard Medical School, 77 Louis Pasteur Ave., Boston, MA 02115. Phone: (617) 432-1960. Fax: (617) 432-2689. E-mail:}ude.dravrah.smh@nimajneb_samoht.

Received 2004 Apr 20; Accepted 2004 Jun 21.

Abstract

The pathway of entry of polyomavirus (Py) has been investigated with glycolipid-deficient C6 cells and added ganglioside GD1a as a specific virus receptor. Unsupplemented C6 cells show a low basal level of infection but become highly infectable by Py following preincubation with the sialic acid-containing ganglioside GD1a (38). Addition of GD1a has no effect on the overall level of virus binding but mediates the internalization and transit of virus to the endoplasmic reticulum (ER). This pathway of entry is cholesterol and caveola dependent and requires intact microtubules as well as a dynamic state of the microfilament system. In contrast to vesicular transport of other cargo via glycolipids, Py particles do not appear to pass through the Golgi apparatus. Colcemid and brefeldin A block transport of the virus to the ER in GD1a-supplemented cells and lead to accumulation of virus in a caveolin-1-containing environment. Several features distinguish the efficient GD1a-mediated pathway of virus uptake from the less-efficient pathway of basal infection in C6 cells.

Abstract

Murine polyomavirus (Py) is a small nonenveloped DNA virus that induces tumors in a wide variety of tissues in the mouse and efficiently transforms cells in culture. The Py virion is composed of 360 copies of the major capsid protein VP1, which assemble into 72 pentamers to form the outer shell of the virus and bind to cell surface sialic acid (8), and 72 copies of the minor proteins VP2-VP3, which form an inner scaffold (2). The ability of the virus to bind to oligosaccharides with terminal α-2,3-linked sialic acid enables the virus to infect multiple cell types in its natural host (3, 6, 7). The crystal structure of VP1 complexed with an oligosaccharide receptor fragment indicates that changes in the contact residues within the binding pocket profoundly affect the ability of sialic acid variants to bind (36, 37). This discrimination between different sialic acid linkages determines the ability of different Py strains to infect cells (14) and to spread in the animal host (3).

The mechanism by which Py is internalized is not fully understood. Early electron microscopic studies indicated that virus particles are taken up into small, uncoated vesicles presumably destined for the endoplasmic reticulum (ER) (13, 18-20, 27, 28). The exact nature of these vesicles has proved to be elusive, however. Previous work indicated that Py is endocytosed by nonclathrin, non-caveola-derived vesicles in a dynamin-independent manner (10, 11), whereas work from other labs implicated a caveola-based vesicle uptake pathway (19, 27). Given the ubiquity of α-2,3-linked sialic acid residues on the cell surface and the broad range of cell types the virus can infect, the possibility exists that Py may attach to multiple receptors and be taken up through more than one endocytic pathway. The effectiveness of different pathways could vary among cell types, depending on the efficiency of delivery to a compartment, presumably the ER, where Py can penetrate the host membrane regardless of the exact pathway of delivery.

Recent efforts in a number of laboratories have focused on the identification of specific molecules bearing sialic acid as Py receptors and on the subsequent pathway of virus internalization and entry in the nucleus. Efforts to identify a specific Py receptor by screening for protective monoclonal antibodies were unsuccessful (3). However, α4β1 integrin, which carries sialic acid moieties, has been reported to function at the postattachment level as a possible secondary receptor (4, 5). C6, a rat glioma cell line that is deficient in the formation of complex gangliosides (34) is poorly infectable by both Py and simian virus 40 (SV40). These cells can be rendered infectable by preincubation with specific gangliosides, GD1a for Py and GM1 for SV40 (38). The present study focuses on the C6 cell system as a model for entry of the whole virus mediated by ganglioside GD1a as a specific receptor.

Acknowledgments

This work has been supported by grant RO1CA082395 from the National Cancer Institute. T.B. is a Virginia and D. K. Ludwig Professor of Cancer Research and Teaching.

We acknowledge fruitful discussions with T. Rapoport and B. Tsai and the technical assistance of J. You.

Acknowledgments

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