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NPXY Motifs in the β1 Integrin Cytoplasmic Tail Are Required for Functional Reovirus Entry

Departments of Microbiology and Immunology,¹ Pediatrics,³ Medicine,⁴ Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine,² Department of Medicine, Veterans Affairs Hospital, Nashville, Tennessee 37232⁵

Received 24 July 2007/ Accepted 15 January 2008

Reovirus cell entry is mediated by attachment to cell surface carbohydrate and junctional adhesion molecule A (JAM-A) and internalization by β1 integrin. The β1 integrin cytoplasmic tail contains two NPXY motifs, which function in recruitment of adaptor proteins and clathrin for endocytosis and serve as sorting signals for internalized cargo. As reovirus infection requires disassembly in the endocytic compartment, we investigated the role of the β1 integrin NPXY motifs in reovirus internalization. In comparison to wild-type cells (β1+/+ cells), reovirus infectivity was significantly reduced in cells expressing mutant β1 integrin in which the NPXY motifs were altered to NPXF (β1+/+Y783F/Y795F cells). However, reovirus displayed equivalent binding and internalization levels following adsorption to β1+/+ cells and β1+/+Y783F/Y795F cells, suggesting that the NPXY motifs are essential for transport of reovirus within the endocytic pathway. Reovirus entry into β1+/+ cells was blocked by chlorpromazine, an inhibitor of clathrin-mediated endocytosis, while entry into β1+/+Y783F/Y795F cells was unaffected. Furthermore, virus was distributed to morphologically distinct endocytic organelles in β1+/+ and β1+/+Y783F/Y795F cells, providing further evidence that the β1 integrin NPXY motifs mediate sorting of reovirus in the endocytic pathway. Thus, NPXY motifs in the β1 integrin cytoplasmic tail are required for functional reovirus entry, which indicates a key role for these sequences in endocytosis of a pathogenic virus.

Published ahead of print on 23 January 2008.