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Journal of Virology, May 1999, p. 4372-4384, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Intracellular Trafficking and Localization of the Pseudorabies Virus Us9 Type II Envelope Protein to Host and Viral Membranes

A. D. Brideau,1 T. del Rio,1 E. J. Wolffe,2 and L. W. Enquist1,*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544,1 and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20895-04452

Received 19 November 1998/Accepted 2 February 1999

The Us9 protein is a phosphorylated membrane protein present in the lipid envelope of pseudorabies virus (PRV) particles in a unique tail-anchored type II membrane topology. In this report, we demonstrate that the steady-state residence of the Us9 protein is in a cellular compartment in or near the trans-Golgi network (TGN). Through internalization assays with an enhanced green fluorescent protein epitope-tagged Us9 protein, we demonstrate that the maintenance of Us9 to the TGN region is a dynamic process involving retrieval of molecules from the cell surface. Deletion analysis of the cytoplasmic tail reveals that an acidic cluster containing putative phosphorylation sites is necessary for the recycling of Us9 from the plasma membrane. The absence of this cluster results in the relocalization of Us9 to the plasma membrane due to a defect in endocytosis. The acidic motif, however, does not contain signals needed to direct the incorporation of Us9 into viral envelopes. In this study, we also investigate the role of a dileucine endocytosis signal in the Us9 cytoplasmic tail in the recycling and retention of Us9 to the TGN region. Site-directed mutagenesis of the dileucine motif results in an increase in Us9 plasma membrane staining and a partial internalization defect.

* Corresponding author. Mailing address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544. Phone: (609) 258-2415. Fax: (609) 258-1035. E-mail: Lenquist{at}molbiol.princeton.edu.

Journal of Virology, May 1999, p. 4372-4384, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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