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Journal of Virology, March 2000, p. 2847-2854, Vol. 74, No. 6
0022-538X/00/$04.00+0

Subcellular Redistribution of Pit-2 Pi Transporter/Amphotropic Leukemia Virus (A-MuLV) Receptor in A-MuLV-Infected NIH 3T3 Fibroblasts: Involvement in Superinfection Interference

Zsolt Jobbagy,1 Susan Garfield,2 Lisa Baptiste,3 Maribeth V. Eiden,3 and Wayne B. Anderson1,*

Laboratory of Cellular Oncology1 and Laboratory of Experimental Carcinogenesis,2 National Cancer Institute, and Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health,3 National Institutes of Health, Bethesda, Maryland 20892

Received 28 July 1999/Accepted 8 December 1999

Amphotropic murine leukemia virus (A-MuLV) utilizes the Pit-2 sodium-dependent phosphate transporter as a cell surface receptor to infect mammalian cells. Previous studies established that infection of cells with A-MuLV resulted in the specific down-modulation of phosphate uptake mediated by Pit-2 and in resistance to superinfection with A-MuLV. To study the mechanisms underlying these phenomena, we constructed plasmids capable of efficiently expressing varepsilon  epitope- and green fluorescent protein (GFP)-tagged human Pit-2 proteins in mammalian cells. Overexpression of varepsilon -epitope-tagged Pit-2 transporters in NIH 3T3 cells resulted in a marked increase in sodium-dependent Pi uptake. This increase in Pi uptake was specifically blocked by A-MuLV infection but not by infection with ecotropic MuLV (E-MuLV) (which utilizes a cationic amino acid transporter, not Pit-2, as a cell surface receptor). These data, together with the finding that the tagged Pit-2 transporters retained their A-MuLV receptor function, indicate that the insertion of epitope tags does not affect either retrovirus receptor or Pi transporter function. The overexpressed epitope-tagged transporters were detected in cell lysates, by Western blot analysis using both varepsilon -epitope- and GFP-specific antibodies as well as with Pit-2 antiserum. Both the epitope- and GFP-tagged transporters showed almost exclusive plasma membrane localization when expressed in NIH 3T3 cells, as determined by laser scanning confocal microscopy. Importantly, when NIH 3T3 cells expressing these proteins were productively infected with A-MuLV, the tagged transporters and receptors were no longer detected in the plasma membrane but rather were localized to a punctate structure within the cytosolic compartment distinct from Golgi, endoplasmic reticulum, endosomes, lysosomes, and mitochondria. The intracellular Pit-2 pool colocalized with the virus in A-MuLV-infected cells. A similar redistribution of the tagged Pit-2 proteins was not observed following infection with E-MuLV, indicating that the redistribution of Pit-2 is not directly attributable to general effects associated with retroviral infection but rather is a specific consequence of A-MuLV-Pit-2 interactions.

* Corresponding author. Mailing address: Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bldg. 37, Room 1E14, 37 Convent Dr., Bethesda, MD 20892. Phone: (301) 496-9247. Fax: (301) 480-0471. E-mail: andersow{at}exchange.nih.gov.

Journal of Virology, March 2000, p. 2847-2854, Vol. 74, No. 6
0022-538X/00/$04.00+0


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