Proteolytic cleavage of HIV-1 GFP-Vpr fusions at novel sites within virions and living cells: concerns for intracellular trafficking studies.
Fluorescent labelling of the highly conserved HIV-1 accessory protein Vpr (Viral Protein R) with GFP or variants thereof has proved a valuable approach to track Vpr and/or HIV-1 subcellular localisation in vivo. Our analysis in transfected mammalian cells expressing GFP-Vpr fusion protein, as well as within virus derived there from, documents site-specific proteolytic cleavage of the GFP-Vpr fusion protein. Western analysis revealed that transfected mammalian cells harbour a C-terminally truncated variant of Vpr in addition to full-length GFP-Vpr. Further, virions derived from these GFP-Vpr expressing cells show protein in which the GFP-tag has been additionally cleaved from the Vpr protein. Endogenous HIV protease (PR) activity was shown to be responsible for the latter, as addition of Saquinavir, a potent PR inhibitor abolished the cleavage. Since many previous studies have relied on imaging the GFP fluorescence of GFP-Vpr, it would appear that the results may not reflect intact GFP-Vpr.
Caly
Leon
L
Department of Biochemistry and Molecular Biology, Monash University, Rm. 204, Building 13D, Clayton, VIC, 3800, Australia.
Jans
David A
DA
Piller
Sabine C
SC
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014771:Virion / Q000378:metabolism*; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1; D049452:Green Fluorescent Proteins